https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=William+EisbrennerProteopedia - User contributions [en]2026-10-03T02:25:10ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=FhuD&diff=1062637FhuD2010-03-31T03:34:39Z<p>William Eisbrenner: Binding pocket</p>
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<div><table style="background-color:#ffffc0" cellpadding="8" width="95%" border="0"><tr><td>Please do NOT make changes to this Sandbox until after April 23, 2010. Sandboxes 151-200 are reserved until then for use by the Chemistry 307 class at UNBC taught by Prof. [[User:Andrea Gorrell|Andrea Gorrell]].</td></tr><br />
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=='''PERIPLASMIC FERRIC SIDEROPHORE BINDING PROTEIN FHUD COMPLEXED WITH COPROGEN (1esz)'''==<br />
<br />
----<br />
<applet load='1esz' size='400' frame='true' align='right' caption='Strucutre of the Periplasmic Ferric Siderophore Binding Protein FhuD complexed with Coprogen as determined by Clarke et al.' /><br />
<br />
==OVERVIEW==<br />
Siderophore-binding proteins can be found in both Gram-positive and Gram-negative bacteria in divisions: hydroxamates, catecholates, and carboxylates. In Escherichia coli. (E. coli) the ATP-binding cassette- type (ABC-type) protein FhuD (part of the “helical backbone” metal receptor superfamily) is a common periplasmic protein which facilitates the transport of a variety of hydoxamate siderophores to the inner membrane-associated proteins FhuB and FhuC. The structure of FhuD is atypical for periplasmic ligand binding protein due to its bilobal mixture of two α/β domains connected by long α-helix. <br />
<br />
==PROTEIN STRUCTURE==<br />
FhuD structure is atypical for periplasmic ligand binding proteins. It is 266 residues in length containing a secondary structure composed of 41% helical (13 helices; 110 residues) and 17% beta sheet (13 strands; 47 residues). It is a bilobal kidney bean shape with approximate dimensions 60 Å ´ 30 Å ´ 40 Å.(ref both) containing two domains which are connected by a 23-residue kinked α-helix. The <scene name='Sandbox_193/N-terminal_domain/1'>N-terminal domain (green)</scene> (residues 27–141) twisted fived-stranded parallel β-sheet with 3-2-1-4-5 linking topology whereas the C-terminal domain (residues 166–288) has a mixed five stranded β-sheet 3-2-1-4-5 linking topology; both are enclosed by α-helices. Between the two domains lies the shallow siderophore <scene name='Sandbox_193/Binding_pocket/1'>binding site (pink)</scene> approximate 10 Å deep (REF) which forms depression or “pocket.” This pocket is lined with hydrophobic residues which side chain residues are able to create stabilizing hydrogen bond with the accepted siderophore. This is large enough to accommodate the hydrophobic orinthyl linkers of the siderophore. Through rearrangements of the residues of the binding pocket and interactions with the iron-hydroxamate centers of the siderophore, recognition can occur with structurally diverse siderophores. The binding diversity is further increased since the siderophore backbones do not interact with the proteins.<br />
<br />
==PROTEIN FUNCTION==<br />
Siderophores function within both gram-positive and gram-negative bacteria to aid in the uptake of iron. They are low molecular weight (500-1000 Da) and can bind with association constraints as high as ten to the power of 50. Let us consider E. coli which produces one siderophore called enterobactin yet can still use many siderophores including hydroxamate type, catecholates type, and citrate. These siderophores require assisted entrance into bacterial cells through uptake systems. In gram negative bacteria, including E. coli, an ATP binding cassette type (ABC) is used which requires several proteins including an outer membrane receptor, periplasmic transport protein and inner membrane proteins. When considering the hydroxamate type siderophore receptors of E.coli, specific outer membrane siderophore binding proteins are used such as FhuA for ferrichrome. After transport in the periplasm, the periplasmic binding protein FhuD is required for the movement of all hydroxamate type siderophores to the cytoplasm and inner membrane proteins FhuB and FhuC. This system allows for distribution of iron to the cell as required by the bacterium. <br />
<br />
==INTERESTING FACTS==<br />
Unlike other periplasmic ligand binding protein (PLBP), FhuD does not have the characteristic fold of a bilobate domain connected by flexible β-strands at the base of the ligand binding pocket. This results in FhuD adopting a novel PLBP structure. As designated by its structure, FhuD binds hydroxamate siderophores into a primarily hydrophobic pocket allowing the assumption that both binding and release do not cause large scale opening/closing. However in the binding pocket, several major ligand binding side chains have been noted to various positions depending on the ligand bound. Due to the ability of siderophore binding uptake systems to allow such a diverse array of siderophore bound molecules, new bacterial growth inhibiting agents may be developed. These agents will be delivered into the bacteria as silent “Trojan Horses” by the bacteria’s own uptake system. <br />
<br />
[[User:Leni Rose|Leni Rose]] 04:57, 13 March 2010 (IST)</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061899T4 RNA ligase 2 (Rnl2)2010-03-28T18:33:36Z<p>William Eisbrenner: </p>
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<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation<ref name="lu">PMID:14962393</ref>. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu"/>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract bacterial host defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore, it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of ''trypanosoma [http://en.wikipedia.org/wiki/Trypanosoma]'', a monophyletic group of unicellular parasitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe 119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys 225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys 227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base <ref name="lu"/>. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys 209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged, it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA <ref name="lu"/>. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positive charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu 99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. In addition, the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2) <ref name="lu"/>. Likewise Glu 204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond (Figure 2). The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His 37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His 37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp 120 and Lys 209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components contributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe 119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pocket of Rnl2. </scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structurally important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser 170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects between 300, 000 to 500, 000 people in rural parts of Central Africa every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms <ref name="lu"/>. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu 34, or His 37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061893T4 RNA ligase 2 (Rnl2)2010-03-28T18:20:34Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation<ref name="lu">PMID:14962393</ref>. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu"/>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract bacterial host defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore, it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of ''trypanosoma [http://en.wikipedia.org/wiki/Trypanosoma]'', a monophyletic group of unicellular parasitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe 119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys 225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys 227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base <ref name="lu"/>. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys 209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged, it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA <ref name="lu"/>. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positive charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu 99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. In addition, the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2) <ref name="lu"/>. Likewise Glu 204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond (Figure 2). The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His 37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His 37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp 120 and Lys 209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components contributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061892T4 RNA ligase 2 (Rnl2)2010-03-28T17:55:47Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation<ref name="lu">PMID:14962393</ref>. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu"/>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract bacterial host defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore, it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of ''trypanosoma [http://en.wikipedia.org/wiki/Trypanosoma]'', a monophyletic group of unicellular parasitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061891T4 RNA ligase 2 (Rnl2)2010-03-28T17:50:46Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation<ref name="lu">PMID:14962393</ref>. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu"/>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract bacterial host defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore, it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of ''trypanosoma [http://en.wikipedia.org/wiki/Trypanosoma]'', a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061890T4 RNA ligase 2 (Rnl2)2010-03-28T17:35:20Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation<ref name="lu">PMID:14962393</ref>. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu"/>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract bacterial host defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore, it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of ''[Trypanosoma]'', a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061889T4 RNA ligase 2 (Rnl2)2010-03-28T17:21:53Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents net positive charged regions while red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=File:Diagram_of_tRNA_repair_by_Rln1.jpg&diff=1061888File:Diagram of tRNA repair by Rln1.jpg2010-03-28T17:18:34Z<p>William Eisbrenner: uploaded a new version of "Image:Diagram of tRNA repair by Rln1.jpg"</p>
<hr />
<div>Figure 1. Mechanism of tRNA ligation of a broken anticodon region catalyzed by Rnl2.</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061886T4 RNA ligase 2 (Rnl2)2010-03-28T16:51:21Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of<scene name='Sandbox_157/T4_rnl2_active_site/24'>alpha helix 6 (lime green)</scene> through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061885T4 RNA ligase 2 (Rnl2)2010-03-28T16:44:18Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene <scene name='Sandbox_157/T4_rnl2_active_site/26'>Asp 120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/27'>Arg 221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/28'> Tyr 5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/29'>Glu 34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1061884T4 RNA ligase 2 (Rnl2)2010-03-28T16:20:22Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name=<scene name='Sandbox_157/T4_rnl2_active_site/25'>(silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple) </scene><ref name="lu"/>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058178T4 RNA ligase 2 (Rnl2)2010-03-19T16:36:03Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='350' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple) </scene><ref name="lu"/>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
<br />
<br />
<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058077T4 RNA ligase 2 (Rnl2)2010-03-19T02:23:14Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s <ref name="lu"/>. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands <ref name="lu"/>. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy <ref name="lu"/>. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding <ref name="lu"/>. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups <ref name="lu"/>. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated) </scene><ref name="lu"/>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme <ref name="lu"/>. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here <ref name="lu"/>. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1) <ref name="lu"/>. [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step <ref name="lu"/>. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well <ref name="lu"/>. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1 <ref name="lu"/>. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step <ref name="lu"/>. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself <ref name="lu"/>. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple) </scene><ref name="lu"/>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains <ref name="lu"/>. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation <ref name="lu"/>. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple) <ref name="lu"/>. <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. If the enzymes behave similar mechanistically, target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37 <ref name="lu"/>. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058072T4 RNA ligase 2 (Rnl2)2010-03-19T02:05:45Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058071T4 RNA ligase 2 (Rnl2)2010-03-19T02:05:22Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
<br />
== Overview: ==<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058025T4 RNA ligase 2 (Rnl2)2010-03-19T01:13:45Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices <ref name="lu">PMID:14962393<br />
</ref>. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site <ref name="lu"/>.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058022T4 RNA ligase 2 (Rnl2)2010-03-19T01:08:39Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE=Sandbox_157/T4_rnl2_active_site/12 }}<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058021T4 RNA ligase 2 (Rnl2)2010-03-19T01:07:38Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | scene=Sandbox_157/T4_rnl2_active_site/13 }}<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058014T4 RNA ligase 2 (Rnl2)2010-03-19T00:54:38Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE='Sandbox_157/T4_rnl2_active_site/12' }}<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1058013T4 RNA ligase 2 (Rnl2)2010-03-19T00:54:09Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
{{STRUCTURE_1s68| PDB=1s68 | SCENE='Sandbox_157/T4_rnl2_active_site/12'}}<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1057959T4 RNA ligase 2 (Rnl2)2010-03-18T20:00:26Z<p>William Eisbrenner: /* Significance of T4 Rnl2 to the medical industry: */</p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:16020726<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1057958T4 RNA ligase 2 (Rnl2)2010-03-18T19:58:12Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease.<ref>PMID:14962393<br />
</ref> Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1057957T4 RNA ligase 2 (Rnl2)2010-03-18T19:56:30Z<p>William Eisbrenner: /* Significance of T4 Rnl2 to the medical industry: */</p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Trypanosomaisis affects betwee 300, 000 to 500, 000 people in rual parts of Central aferica every year in which almost all cases are fatal and currently no effective treatment method exists for the disease. Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1057956T4 RNA ligase 2 (Rnl2)2010-03-18T19:52:16Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is necessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly or indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine monophosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surrounding AMP is positively charged it may contribute to the affinity of the enzyme for the negatively charged 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophilic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It accomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, in particular its active site and binding sites, an understanding of this protein's structure in relation to its function can give further insight as to how the REL's structure may relate to their function. In if the enzymes behave similarly mechanistically target pharmaceuticals designed to inhibit the Rnl2 active site or binding sites may also serve as potential inhibitors against RELs. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed to mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1055669T4 RNA ligase 2 (Rnl2)2010-03-11T20:03:18Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site.<br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1055668T4 RNA ligase 2 (Rnl2)2010-03-11T19:58:58Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation.<ref>PMID:14962393<br />
</ref> Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices.<ref>PMID:14962393<br />
</ref> The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site.<ref>PMID:14962393<br />
</ref> <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significance of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <ref>PMID:14962393<br />
</ref><br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053873T4 RNA ligase 2 (Rnl2)2010-03-09T20:39:04Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below (Figure 1). [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <ref>PMID:14962393<br />
</ref><br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053872T4 RNA ligase 2 (Rnl2)2010-03-09T20:38:00Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. [[Image:Diagram of tRNA repair by Rln1.jpg |left| thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <ref>PMID:14962393<br />
</ref><br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053871T4 RNA ligase 2 (Rnl2)2010-03-09T20:33:31Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. [[Image:Diagram of tRNA repair by Rln1.jpg | thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <ref>PMID:14962393<br />
</ref><br />
----<br />
<br />
== References: ==<br />
<br />
<references/></div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053865T4 RNA ligase 2 (Rnl2)2010-03-09T20:24:19Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. [[Image:Diagram of tRNA repair by Rln1.jpg | thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339<br />
<ref group="xtra">PMID:14962393</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053859T4 RNA ligase 2 (Rnl2)2010-03-09T20:10:07Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. [[Image:Diagram of tRNA repair by Rln1.jpg | thumb|alt=Diagram of tRNA repari by Rnl2. | Figure 1. Mechanism of tRNA ligation of anticodon region by T4 RNA ligase 2 (Rnl2) catalyst.]] First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 2. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=File:Diagram_of_tRNA_repair_by_Rln1.jpg&diff=1053856File:Diagram of tRNA repair by Rln1.jpg2010-03-09T19:55:04Z<p>William Eisbrenner: Figure 1. Mechanism of tRNA ligation of a broken anticodon region catalyzed by Rnl2.</p>
<hr />
<div>Figure 1. Mechanism of tRNA ligation of a broken anticodon region catalyzed by Rnl2.</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1053829T4 RNA ligase 2 (Rnl2)2010-03-09T15:39:06Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it has been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1050641T4 RNA ligase 2 (Rnl2)2010-02-26T20:48:36Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/><br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1050640T4 RNA ligase 2 (Rnl2)2010-02-26T20:42:01Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1050639T4 RNA ligase 2 (Rnl2)2010-02-26T20:35:02Z<p>William Eisbrenner: </p>
<hr />
<div>== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
<br />
<br />
== Overview: ==<br />
<br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
<br />
<br />
== Protein Function: ==<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
<br />
<br />
== Structural Components Contributing to the Active Site: ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
<br />
<br />
== Significants of T4 Rnl2 to the medical industry: ==<br />
<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
== References: ==<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1050638T4 RNA ligase 2 (Rnl2)2010-02-26T20:29:25Z<p>William Eisbrenner: </p>
<hr />
<div><br />
== T4 RNA ligase 2 (Rnl2) ==<br />
<br />
<applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/><br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
----<br />
<br />
'''Significants T4 Rnl2 to the medical industry''':<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048704T4 RNA ligase 2 (Rnl2)2010-02-18T20:37:58Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
----<br />
<br />
'''Significants T4 Rnl2 to the medical industry''':<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048703T4 RNA ligase 2 (Rnl2)2010-02-18T20:37:22Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
----<br />
<br />
'''Significants T4 Rnl2 to the medical industry'''<br />
<br />
Because of the several similarities Rnl2 shares with REL-1 and other RELs, an understanding of this proteins structure in relation to its function can give further insight to how the RELs structure may relate to their function. Since the RELs have been found to be essential to trypanosomiasis and leishmaniasis, mentioned earlier, they have become prime targets for drugs against these microorganisms. Such drugs can be designed mimic the substrate, bind to impair the AMP binding pocket, or bind to other surface residues which are essential to the function of the protein such as Glu34, or His37. <br />
----<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048695T4 RNA ligase 2 (Rnl2)2010-02-18T19:38:52Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
----<br />
<br />
'''Significants T4 Rnl2 to the medical industry'''<br />
<br />
<br />
----<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048694T4 RNA ligase 2 (Rnl2)2010-02-18T19:32:34Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <scene name='Sandbox_157/T4_rnl2_active_site/24'>Glu34 (dark red)</scene> has also been found to be structually important, although not making direct contact with AMP, as it functions to make sure that the loop containing the lysine nucleophile is in proper conformation for adenylation. It acomplishes this structural function by tethering the backside of the motif I loop (green) to the start of alpha helix 6 (lime green) through a Hydrogen bond to Ser170 (dark purple). <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048693T4 RNA ligase 2 (Rnl2)2010-02-18T19:08:06Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> <scene name='Sandbox_157/T4_rnl2_active_site/23'>Click here</scene> to see H-bonding patterns between side chains. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048692T4 RNA ligase 2 (Rnl2)2010-02-18T19:05:33Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These <scene name='Sandbox_157/T4_rnl2_active_site/22'>structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2</scene> Click here to see H-bonds between side chains. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048691T4 RNA ligase 2 (Rnl2)2010-02-18T18:30:55Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to <scene name='Sandbox_157/T4_rnl2_active_site/21'>Tyr5 (dark purple)</scene>. These structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048690T4 RNA ligase 2 (Rnl2)2010-02-18T18:23:48Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to <scene name='Sandbox_157/T4_rnl2_active_site/20'>Arg221 (dark green)</scene> and a hydrogen bond to Tyr5. These structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048689T4 RNA ligase 2 (Rnl2)2010-02-18T18:17:45Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''': <applet load='1s68' size='400' frame='true' align='left' caption='Structural components cotributing to the Rnl2 active site' scene='Sandbox_157/T4_rnl2_active_site/13'/>T4 RNA ligase 2 (Rnl2):<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance <scene name='Sandbox_157/T4_rnl2_active_site/19'>Asp120 (silver)</scene> of motif IIIa (pink), located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to Arg221 and a hydrogen bond to Tyr5. These structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048687T4 RNA ligase 2 (Rnl2)2010-02-18T17:59:40Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''':<br />
<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. For instance Asp120 of motif IIIa, located next to Phe119 (see above for function), serves as a focal point of hydrophillic contacts which include a bidentate salt bridge to Arg221 and a hydrogen bond to Tyr5. These structural components tether 4 beta strands that make up the nucleotide binding pockets of Rnl2. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048685T4 RNA ligase 2 (Rnl2)2010-02-18T17:34:10Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
'''Overview''': <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
'''Protein function''':<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. It has also been found that residues Asp120 and Lys209 are also both essential for Rnl2 activity even though they are not in direct association with AMP itself. <br />
----<br />
'''Structural components contributing to the active site''':<br />
Above the components of the actual active site were discussed in relation to the function of the protein, however there are several indirect structural components which are linked to maintaining active site structure as well as aiding in conformational changes while it carries out its function. <br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048623T4 RNA ligase 2 (Rnl2)2010-02-18T01:54:18Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
Overview: <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
Protein function:<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His37 (light green)</scene> in motif 1. Ho et al. hypothesized that His37 sets the boundaries of the binding site for the 3' OH RNA strand which acts as a nucleophile in this step and hence is necessary for catalyzing this step. <br />
----<br />
Structural components contributing to the active site:<br />
----<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrennerhttps://proteopedia.org/index.php?title=T4_RNA_ligase_2_(Rnl2)&diff=1048618T4 RNA ligase 2 (Rnl2)2010-02-18T01:24:55Z<p>William Eisbrenner: </p>
<hr />
<div><applet load='1s68' size='400' frame='true' align='right' caption='T4 Rnl2' scene='Sandbox_157/T4_rnl2_active_site/12'/>T4 RNA ligase 2 (Rnl2):<br />
----<br />
<br />
Overview: <br />
<br />
T4 RNA ligase 2 (Rnl2) (1-249) is a T4 bacteriophage ligase, which functions to counter bacterial defence by repairing broken anticodon loops of tRNA coding for lysine, caused by PrrC, which is nessessary for viral replication and proliferation. Note that the structure shown is based on the truncated enzyme form (1-249) where the total chain length of Rnl2 is 334 residues. <br />
The overall structure, at 100K, pH 8.5, consists of a single chain polymer (233 residues + 15 His tag residues) composed of 2, 6 antiparalel stranded, beta sheets and 7 alpha helices. The structure contains an adenosine monophosphate ligand in its active site. The enzyme contains 5 nucleotidyl transferase motifs,<scene name='Sandbox_157/T4_rnl2_active_site/13'>I, III, IIIa, IV and V</scene> (green, blue, pink, light blue, red) which are all involved either directly for indirectly with the function of the active site. <br />
----<br />
<br />
Protein function:<br />
<br />
Although this protein is designed to counteract the bacterial host’s defence mechanisms against the virus, its activity as a ligase is analogous to tRNA splicing (introns removed from anticodon loop) and RNA editing by several different kinds of RNA editing ligases (REL)s. Therefore it have been grouped into a subfamily of RNA ligases including REL-1 which is essential for the survival of Trypanosoma, a monophyletic group of unicellular paracitic flagella protozoa that causes sleeping sickness. Another commonality shared between RNA ligases as well as DNA ligases and capping enzymes is the fact that the mechanism by which they function involves the formation of a covalent enzyme-(lysyl-N)-NMP intermediate prior to the formation of the phophodiester bond between 5’ and 3’ ends of RNA strands. Hence these groups have been classified as a super family of enzymes, characteristic of the above intermediate. <br />
<br />
The mechanism for the formation of this intermediate and the phosphodiester bond resulting in RNA strand repair is outlined below. First ATP reacts with the active site of Rln2. Adenosine mono phosphate <scene name='Sandbox_157/T4_rnl2_active_site/3'>(AMP)</scene> binds to the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site (light green)</scene> accompanied by the release of inorganic phosphate which provides the energy. The adenylate binds at the bottom of the <scene name='Sandbox_157/T4_rnl2_active_site/1'>active site</scene> (motif 1) where it is squished between the aromatic rings of <scene name='Sandbox_157/T4_rnl2_active_site/4'>Phe119 (light blue)</scene> of motif IIIa, <scene name='Sandbox_157/T4_rnl2_active_site/5'>Val 207 (purple)</scene> of motif III and <scene name='Sandbox_157/T4_rnl2_active_site/6'>Lys 35 (yellow)</scene> of motif I. <scene name='Sandbox_157/T4_rnl2_active_site/14'>Lys225 (orange)</scene> and <scene name='Sandbox_157/T4_rnl2_active_site/15'>Lys227 (peach)</scene> of motif V, which are essential for Rln2 activity, are also involved in coordinating the AMP phosphate after binding. During ligase adenylation the alpha phosphate of ATP is thought to be stabilized by Lys 225 and 227 prior to release of the other phosphate groups. The specificity of Rln2 for ATP as opposed to other NTP substrates in this step are thought to be attributed to hydrogen bonding between backbone residues and the adenine base. Such bonds occur between:<scene name='Sandbox_157/T4_rnl2_active_site/7'>N7 (on AMP) and the backbone amide of Ile36 (dark green),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/8'>the exocyclic 6-amino group (N6) and main chain carbonyl (O) of Glu34 (dark purple),</scene> <scene name='Sandbox_157/T4_rnl2_active_site/9'>N1 and Lys209 (dark red) (water (white circles) mediated)</scene>. After Binding AMP, the Rnl2 transfers AMP to the 5' phosphate of the RNA strand. The binding of Rnl2 to RNA, required for this step, can be partially attributed to the electrostatic structure of the enzyme. Since the active site surface surounding AMP is possitively charged it may contribute to the affinity of the enzyme for negatively charge 5' phosphate of pRNA. Note that all interactions between pRNA and Rnl2 also require the essential C-terminal domain which is not shown in the crystalized structure here. This may be due to the essential positioning requirements for the placement of ATP in correct close proximity and orientation to the 5' phosphate for the next step (Figure 1). [[Image:Rnl2 Electorstatic potential.jpg | thumb|alt=T4 Rnl2.| Figure 1. Electrostatic potential of Rnl2 where blue represents positive charges and red represents net negative charged regions. Majority of the surfaces have a net negative charge while positve charges are localized around AMP in the surrounding active site.]] The interactions between the ribose sugar and the protein back bone are all thought to contribute to this adenyltransferase function as well as overall pRNA ligation, where the interaction between the<scene name='Sandbox_157/T4_rnl2_active_site/16'> 2' oxygen of the ribose sugar and Glu99 (teal)</scene> is thought to be most essential to this step. As well the <scene name='Sandbox_157/T4_rnl2_active_site/17'>Arg55 (dark blue) guanidinium group (NH2, NE, NH1)</scene>, which coordinates the 3' O of the ribose sugar, is thought to interact with the 5'-PO<sub>4</sub> of RNA during this adenylation step as well as during initial adenylation of the enzyme by ATP (Figure 2). Likewise Glu204 has been found to be essential to both of these steps as well. The <scene name='Sandbox_157/T4_rnl2_active_site/10'>π stacking between Phe119 and the adenylate base</scene> is thought to be critical for the next step; transfer of the phosphate from the 5’ end of the RNA strand to the 3’ OH end of the RNA strand to form the phosphodiester bond Figure 2. The actual catalytic action of this step however, is thought to be carried out by <scene name='Sandbox_157/T4_rnl2_active_site/18'>His 37 (light green)</scene> in motif 1<br />
----<br />
<br />
Current Refences: Ho et al. 2004 Structure and Mechanism of RNA Ligase. 12:327-339</div>William Eisbrenner