Proteopedia - User contributions [en]

Lsm

2013-04-05T03:26:15Z

Kelly Hrywkiw:

<StructureSection load='3pgw' size='600' side='right' scene='Sandbox_502/U1_sm_ring/2' caption='[[3pgw]]'>
='''Lsm Protein Structure'''=

by Kelly Hrywkiw
__TOC__

=Introduction=

Sm-like ([http://en.wikipedia.org/wiki/LSm Lsm]) proteins most closely resemble Sm proteins, both of which are found in the three domains of life <ref name ="wu">PMID:22615807</ref>. Sm proteins play a large role in [http://en.wikipedia.org/wiki/Spliceosome spliceosome] biogenesis through mediating U1, U2, U4, U5, and U6 [http://en.wikipedia.org/wiki/SnRNP snRNP assembly]<ref name ="he">PMID:10801455</ref>. The <scene name='Sandbox_502/U1_sm_ring/2'>Sm ring</scene> of proteins can be broken down into seven specific proteins (SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG in humans) all of which share a conserved Sm motif that is also found in the Lsm proteins<ref name ="he"/><ref name ="naidoo"/>. Eukaryotes have 16 or more Lsm proteins encoded in their genome, in contrast archaeal species have only one to three <ref name ="naidoo">PMID:18329667</ref>. A total of nine specific Lsm proteins are found in yeast (Lsm1-Lsm9). The Lsm proteins 2-7 most closely resemble Sm proteins D1-G, where Lsm 1 and 8 most closely resemble the SmB protein <ref name ="he"/>. Lsm9 does not appear to resemble any of the Sm proteins, although there have been some related structures found in the archaeal genome <ref name ="he"/>. Several studies have shown that the Sm proteins form into seven membered rings which bind to the Sm binding site, a U rich sequence found in all but U6 snRNA<ref name ="he"/>. Lsm proteins can form homomeric rings of heptamers, hexamers, or octamers<ref name ="wu"/>. In addition they have been found to predominately associate into three complexes: Lsm2-8, Lsm1-7, and Lsm2-7 <ref name ="wu"/>. The exact functionality of these complexes is in either [http://en.wikipedia.org/wiki/RNA_splicing pre-mRNA splicing], [http://en.wikipedia.org/wiki/Messenger_RNA#Degradation mRNA decay] or other roles, and is dictated by their composition, structure, and cellular location <ref name ="wu"/><ref name ="he"/>.

==The Different Lsm Complexes==

Evidence suggests that there are two main Lsm complexes, Lsm1-7 which is associated with mRNA decay, and Lsm 2-8 which is associated with pre-mRNA splicing<ref name ="wu"/><ref name ="he"/>. The first piece of evidence to suggest different roles is the cellular localization of the different Lsm complexes<ref name ="wu"/>. For example, Lsm1 is predominantly cytoplasmic (where mRNA degradation takes place), as is the Lsm1-7 complex<ref name ="wu"/>. The Lsm complex 2-8 is most likely nuclear because that is the location of U6 snRNP assembly <ref name ="pannone">PMID:10898971</ref>. In addition, mutation experiments have shown that while Lsm2 to Lsm7 mutants have altered mRNA decay and splicing function, Lsm1 and Lsm8 mutants only have altered mRNA decay and pre-mRNA splicing function respectively <ref name ="he"/>. Further evidence is found in [http://en.wikipedia.org/wiki/Immunoprecipitation immunopercipitation] experiments. For example, while Lsm2 to Lsm7 co-immunopercipitate with both U6 snRNA and with mRNA decay factors, Lsm1 and Lsm8 only co-immunopercipitate mRNA degradation factors and U6 snRNA respectively <ref name ="he"/>. Due to the difference in functionality of either Lsm1 or Lsm8 it is interesting to note that Lsm1 and Lsm8 are both closely related structurally to the SmB protein<ref name ="he"/>.

==Role in Pre-mRNA splicing==

The processing of [http://en.wikipedia.org/wiki/Pre-mRNA pre-mRNA] takes place through the use of a large dynamic machine known as the spliceosome, through which [http://en.wikipedia.org/wiki/Intron introns] are removed and [http://en.wikipedia.org/wiki/Exon exons] are spliced together to create a mature [http://en.wikipedia.org/wiki/MRNA mRNA]<ref name ="architecture of"/><ref name ="structure and function">PMID:19000813</ref>. The spliceosome is comprised of five [http://en.wikipedia.org/wiki/SnRNA snRNA] molecules (snRNAs U1, U2, U4, U5, and U6) and over one hundred associated proteins<ref name ="architecture of"/><ref name ="structure and function"/>. Assembly of the spliceosome is thought to take place in a stepwise manner around the pre-mRNA transcript<ref name ="architecture of"/><ref name ="structure and function"/>. The first step involves recognition of the 5’ splice site by U1 snRNP, followed by recognition of the branch point sequence by U2 snRNP<ref name ="architecture of"/><ref name ="structure and function"/>. From this point the remaining snRNPs U4, U5, and U6 join as a preformed tri-snNRP<ref name ="architecture of"/><ref name ="structure and function"/>. Together the five snRNPs form the precatalytic spliceosome which must undergo a series of changes before it can actively splice<ref name ="structural evidence">PMID:19525970</ref><ref name ="structure and function"/><ref name ="common design">PMID:19525970</ref><ref name ="architecture of">PMID:22471593</ref>.

The Lsm complex 2-8 is involved in pre-mRNA splicing through association with the 3’terminal poly(U) tract of U6 snRNA <ref name="pannone"/>. U6 snRNP is different from the other snRNPs because it is completely assembled in the [http://en.wikipedia.org/wiki/Cell_nucleus nucleus], whereas the other snRNAs first travel to the [http://en.wikipedia.org/wiki/Cytoplasm cytoplasm] <ref name ="pannone"/><ref name ="architecture of"/>. While the exact mechanism by which the Lsm2-8 complex acts is unclear, it is thought that it provides stability and function to the U6 snRNP<ref name ="he"/>. For example, several experiments using mutants with point mutations in the Lsm proteins 2-8 have shown defects in splicing that correlate with low levels of U6 snRNA <ref name ="he"/>. It may also play a role in the various rearrangements that are necessary throughout the splicing cycle, and has been shown to be important in the assembly of U4-U6 di snRNP and U4-U5/U6 tri snRNP <ref name ="he"/>. An interesting difference between the Sm and Lsm proteins is that in order to assemble the Sm ring RNA must be present, yet this is not a requirement in Lsm ring assembly <ref name ="pannone"/>. Overall, there is significant evidence to suggest that the Lsm proteins 2-8 play a key role in spliceosome biogenesis and architecture.

==Role in mRNA decay==

In yeast degradation of mRNA takes place by first shortening the poly(A) tail then the removing the 5’cap by [[4a53|Dcp1]], a decapping protein <ref name ="pannone"/>. The major exoribonuclease in mRNA decay ([[2y35|Xrn1]]) then quickly degrades the decapped mRNA <ref name ="pannone"/>. Interestingly, when the Lsm 1-7 complex is purified Xrn1, Dcp1, Mrt1 (an additional protein associated with mRNA decapping), and mRNA co-immunopercipitate <ref name ="pannone"/>. However, it is not only Lsm 1 that plays a role in mRNA decay. Lsm mutants of the 2-7 proteins have increased amounts of capped, deadenylated mRNAs <ref name ="pannone"/>. There are two postulated functions of the Lsm1-7 complex in mRNA decay. The first suggests that the Lsm ring binds to the mRNA first then recruits the Dcp1, the second function may be to facilitate rearrangements of the mRNP complex to allow decapping enzymes access to the 5’cap <ref name ="he"/>. Lsm proteins therefore do not only appear to play a role in creating functional mRNA, but also in degradation of mature mRNA.

==Other roles of Lsm proteins==

A third complex of Lsm proteins (Lsm2-7) is found in the nucleoli of Saccharomyces cerecisiae <ref name ="wu"/>. It is believed to play a role in the function or biogenesis of snoRNAs. Other potential roles of the Lsm proteins include processing of tRNAs, snoRNAs, and rRNAs, histone mRNA decapping, miRNA biogenesis, and maturation and/or stabilization of nascent RNA polymerase III transcripts <ref name ="wu"/><ref name ="he"/>.

=Structure of Lsm proteins=

<scene name='Sandbox_502/Splsm3start/1'>Sm and Lsm proteins both exhibit the Sm motif</scene>, which consist of an <scene name='Sandbox_502/Splsm3_alpha/2'>N-terminal</scene> [http://en.wikipedia.org/wiki/Alpha_helix α-helix] proceeded by a twisted <scene name='Sandbox_502/Splsm3_beta/2'>five stranded </scene>[http://en.wikipedia.org/wiki/Beta_sheet β-sheet] <ref name ="wu"/><ref name ="naidoo"/>. <scene name='Sandbox_502/Sclsm3/3'>Loop L4</scene>, located between stands <scene name='Sandbox_502/Sclsm3b3/2'>β3</scene> and <scene name='Sandbox_502/Sclsm3b4/1'>β4</scene> of the β sheet, varies between 3 to 30 residues in length across the different Lsm proteins <ref name ="naidoo"/>. The β-sheet encloses a set of hydrophobic residues <ref name ="naidoo"/>. When the Lsm ring is assembled the hydrophobic region spreads into the now adjacent Lsm protein monomers <ref name ="naidoo"/>. When assembled into the ring between each subunit there are hydrogen bonds formed between <scene name='Sandbox_502/Sclsm3b4ofa/2'>β4 of one subunit</scene> and <scene name='Sandbox_502/Sclsm3b4ofb/2'>β5 of the neighboring subunit</scene> <ref name ="naidoo"/>. These interactions provided the Lsm ring with enough contacts to make a very stable structure <ref name ="naidoo"/>. There are two sides to the ring, the helix face and the loop face, found on <scene name='Sandbox_502/Splsm3helixface/4'>opposite sides</scene> of the ring <ref name ="wu"/>. It has been postulated that a U-rich RNA may bind to the inner portion of the helix face, and take part in hydrogen bonding interactions with residues located on loops 3 and 5, as well as potentially pass through the <scene name='Sandbox_502/Splsm3pore/2'>pore</scene> itself <ref name ="naidoo"/>.

It is possible for single Lsm proteins to form homomeric heptamers, hexamers, or octamers, as well as the Lsm1-7 or 2-8 hexamers. In addition, Lsm proteins have been found to form higher order quaternary structures during the crystallization process <ref name ="naidoo"/><ref name ="wu"/>. These interactions are formed between helix-helix faces, loop-loop faces, and helix-loop faces <ref name ="naidoo"/>. The crystal structures available for analysis do not consist of full Lsm1-7 or Lsm2-8 complexes. However, the Lsm3 monomer, the N-terminal region of Lsm4, and an Lsm complex Lsm5-7 have been crystallized.

==Lsm 3==

The Lsm3 protein had been crystalized from Schizosaccharomyces pombe and from Saccharomyces cerevisiae, hereby referred to as SpLsm3 and ScLsm3 at 2.7Å and 2.5Å respectively.

===ScLsm3===

The ScLsm3 crystal structure takes the form of a ring composed of eight monomeric subunits. Each monomer contains the Sm motif containing the N-terminal α-helix (pro4-leu10) and the curved β-sheet (Glu14-Ser77). The stands β3 and β4 are long, which causes loop L4 residues to stick out and twist away from the main body of the ring. The only other Sm/Lsm protein to exhibit this is the human Sm protein SmB. Between each of the subunits there are hydrogen interactions between the C-terminal region of β4 and the neighboring β5. In addition, there are <scene name='Sandbox_502/Sclsm3residues/1'>hydrophobic residues</scene> buried at this interface, which include Phe67, Ile68, Thr74, and Ile76. The overall ring structure is approximately 75Å wide, 50Å thick. The pore is approximately 20Å at the helix face and 25Å at the loop face. These measurements are greater than those of six or seven membered Lsm rings <ref name ="naidoo"/>.

===SpLsm3===

As in ScLsm3, <scene name='Sandbox_502/Splsm3/4'>SpLsm3</scene> exhibits the sm motif containing an N-terminal α-helix (residues 10-17) and a curved β-sheet (residues 19-89). However, rather than forming an octomeric ring structure it formed a heptameric ring structure in crystallization experiments. SpLsm3 monomers interact through the same β4-β5 pairing as in ScLsm3. The overall ring is 61.5Å wide, 31Å thick, where the pore is approximately 20.7Å wide. In this crystal structure loop four is distorted<ref name ="wu"/>.

==Lsm 4==

The <scene name='Sandbox_502/Splsm4/2'>Lsm4 crystal structure</scene> contains a trimer of the Lsm4 monomers. It contains the Sm motif consisting of an α-helix (distorted) and a β-sheet formed by five antiparallel stands (residues 14-70)<ref name ="wu"/>.

==Lsm 5/6/7==
<scene name='Sandbox_502/Splsm657m/1'>Lsm 5/6/7</scene>

A 2.5Å resolution structure of Lsm5, Lsm6 and Lsm7 has been determined where the crystal contains two hexameric Lsm657-657 rings. <scene name='Sandbox_502/Splsm657m5/1'>Lsm5</scene> is located between <scene name='Sandbox_502/Splsm657m6/2'>Lsm6</scene> and <scene name='Sandbox_502/Splsm657m7/1'>Lsm7</scene> which analogous to their Sm counters parts. In the hexameric ring each subunit interacts in the same manner as the other Lsm proteins (ie through the β4 stand of one subunit to the β5 strand of the other) to form a continuous β-sheet through the whole ring. Each of the Lsm proteins exhibits the Sm motif with very small differences seen between them <ref name ="mund">PMID:22001694</ref>.

===Role in RNA binding===

With respect to the role of Lsm proteins binding to RNA substrates, the pore of the Lsm657-657 ring is positively charged, which would confer to interactions with negatively charged RNA. The Sm ring of Archaeoglobus fulgidus in complex with polyU RNA shows that each of the Sm proteins interacts with one base of RNA through residues in loops 3 and 5, and that the RNA is passed through the pore. Due to the fact that the residues between the Sm and Lsm proteins are fairly conserved it is possible that the Lsm proteins act through a similar mechanism. Two main differences can be seen however. There should be a canonical arginine or lysine in loop five of Lsm5 that forms a hydrogen bond to a base in the RNA, yet there is an asparagine present. In addition, a canonical aromatic residue that provides stacking interactions with an RNA base should be found in loop three of Lsm7, however there is a leucine present instead. While these differences prevent one from applying the RNA-protein interactions of Sm proteins to Lsm proteins, future studies may elucidate the exact mechanism <ref name ="mund">PMID:22001694</ref>.

</StructureSection>
=Additional Resources=

*[http://www.rcsb.org/pdb/explore/explore.do?structureId=3BW1 Crystal structure of homomeric yeast Lsm3 exhibiting novel octameric ring organisation, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=4EMK Crystal structure of SpLsm5/6/7, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=4EMG Crystal structure of SpLsm3, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=4EMH Crystal structure of SpLsm4, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=3SWN Structure of the LSm657 Complex: An Assembly Intermediate of the LSm1 7 and LSm2 8 Rings, in the RCSB Protein Data Bank]

=References=
<References/>

HDim1/U5-15kD

2012-08-24T03:24:42Z

Kelly Hrywkiw: /* Reduced Dominant Negative Form (hDim1-128) */

== Structure of the hDim1/U5-15kD Protein ==

by Kelly Hrywkiw
{{STRUCTURE_1qgv | PDB=1qgv | SCENE= }}
__TOC__

=Introduction=

The protein Dim1 goes by many names, hDim1/U5-15kD in humans, Dim1p in [http://en.wikipedia.org/wiki/Schizosaccharomyces_pombe ''Schizosaccharomyces pombe''] (''S. pombe''), and Dib1p/Snu16p in [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae ''Saccharomyces cerevisiae''] (''S. cerevisiae'')<ref name ="zhang 1999">PMID:11015569</ref><ref name ="Reuter">PMID:10610776</ref><ref name ="Zhang 2003">PMID:12911302</ref>. Regardless of the name it is extraordinarily well conserved across the eukaryotic kingdom sharing 79% sequence identity between the human and ''S. pombe'' orthologs, and 66% sequence identity between the human and ''S. cerevisiae'' orthologs<ref name ="Zhang 2003"/><ref name ="Reuter"/>. Dim1 exhibits a [[Thioredoxin|thioredoxin]] like domain core, and is thought to play a multifunctional role in the [http://en.wikipedia.org/wiki/Spliceosome spliceosome] – the [http://en.wikipedia.org/wiki/Pre-mRNA pre-mRNA] splicing machine (Zhang 2003)<ref name ="Zhang 2003"/><ref name ="Reuter"/>.

==Pre-mRNA Splicing==

Pre-mRNA contains [http://en.wikipedia.org/wiki/Exons exons] (protein coding regions) separated by [http://en.wikipedia.org/wiki/Introns introns] (non-coding regions)<ref name ="Sperling">PMID:19000813</ref>. The spliceosome catalyzes the removal of the introns and the ligation of the remaining exons to create mature mRNA. This reaction takes place through two [http://en.wikipedia.org/wiki/Transesterification transesterification] reactions catalyzed by specific portions of the spliceosome<ref name ="Jurica">PMID:12431437</ref>. The overall structure of the spliceosome is highly dynamic and consist of five integral complexes known as small nuclear ribonucleoprotein particles [http://en.wikipedia.org/wiki/SnRNP (snRNPs)] U1, U2, U4, U5 and U6 <ref name ="Sperling"/>. Each snRNP consist of a uridine rich small nuclear RNA [http://en.wikipedia.org/wiki/SnRNA (snRNA)], seven sm or sm-like [[Lsm|(Lsm)]] proteins, and several additional proteins<ref name ="van der Feltz">PMID:22471593</ref>.

It has been proposed that the snRNPs associate in a stepwise manner <ref name="Sperling"/>. The first step involves U1 snRNP recognizing the 5’ splice site, followed by an ATP dependent step in which U2 associates with the branch point <ref name ="Sperling"/><ref name ="van der Feltz"/>. The tri-snRNP comprised of U4/U6 U5 then binds, and through a series of rearrangements U6 replaces U1 at the 5’ splice site resulting in an activated complex that can perform the first of two transesterification reactions <ref name ="Sperling"/><ref name ="van der Feltz"/>. U1 and U4 then dissociate to yield the second active complex which completes the second reaction <ref name ="Sperling"/><ref name ="van der Feltz"/>. The remaining snRNPs then dissociate. It should be noted that these processive structural rearrangements would not be possible without the presence of ATP <ref name ="van der Feltz"/>.

==Role of Dim1 in Splicing==

While the role of Dim1 in splicing is not well understood is has been shown, in multiple studies, to be a constituent of the tri-snRNP, and to strongly associate specifically with the U5 snRNP <ref name ="Zhang 2003"/><ref name ="Reuter"/>. It is also required for the splicing of a non pre-mRNA, U3 RNA<ref name ="Zhang 2003"/>. Furthermore, hDim1 has been shown to interact with the proteins hnRNP F and hnRNP H’, which enhance tissue specific pre-mRNA splicing, as well as Npw38/PQBP-1 which can bind poly(rG) and co-localize with splicing co-activators when co-expressed with hDim1<ref name ="Zhang 2003"/>. hDim1 also contains RNA recognition motif-like sequences, which suggest that it could directly interact with poly(rG)<ref name ="Zhang 2003"/>. Dib1p strongly interacts with Prp6, a protein required for tri-snRNP accumulation. Overall, this suggests that Dim1 plays a multifaceted role in splicing biogenesis<ref name ="Zhang 2003"/><ref name ="Reuter"/>.

=Structure=

Two structures of Dim1 have been solved, an oxidized full length form and a reduced dominant negative form. The oxidized full length form of Dim1 consists of 142 amino acids and its overall structure adopts a thioredoxin like core domain coupled with a C-terminal extension<ref name ="Reuter"/>. The reduced dominant negative form contains 128 amino acids, where the C-terminal extension containing residues 129-142 have been removed<ref name ="Zhang 2003"/>.

==Oxidized Full Length Form (hDim1)==

<Structure load='1qgv' size='300' frame='true' align='left' caption='Figure 1: ' scene='Sandbox_502/Hdim1_start_scene/2'/>

The thioredoxin-like fold of <scene name='Sandbox_502/Hdim1_start_scene/2'>hDim1</scene> follows the arrangement of a five stranded β-sheet, consisting of parallel and antiparallel stands, surrounded by three α-helices, where the loop between β4 and α3 could not be resolved. When compared to human thioredoxin there are a total of 37 additional residues in hDim1, which result in several structural differences. For example, the N-terminus is extended by three residues, in the α2-β2 loop one residue is inserted, after β4 nine are inserted, before the α2 helix two are inserted, and <scene name='Sandbox_502/Hdim1_c-terminal_extension/1'>22 extend the C-terminus</scene>. This results in an altered structure where β4 and β5 appear to be pulled away from the β-sheet leaving a <scene name='Sandbox_502/Hdim1cleft/1'>cleft</scene> between β3 and β4<ref name ="Reuter"/>.

In thioredoxin there is an N-terminal Cys-X-X-Cys motif which forms a functional [http://en.wikipedia.org/wiki/Disulfide_bond disulfide bond], in hDim1 there is an <scene name='Sandbox_502/Hdim1asp-x-x-cys/1'>Asp-X-X-Cys motif</scene>. Interestingly, in hDim1 the Cys residue still participates in the formation of a disulfide bond with a residue located in β3 (Cys79) resulting in a <scene name='Sandbox_502/Hdim1disulfide_bond/1'>disulfide bond</scene> located in a similar area as that in thioredoxin. Through a series of experiments it has been shown that hDim1 does not appear to participate in redox or protein disulfide isomerase activity as seen in thioredoxin. However, it still may be possible that hDim1 forms disulfide bonds with other spliceosomal proteins, or exhibits [http://en.wikipedia.org/wiki/Peroxiredoxin peroxiredoxin]-like activity<ref name ="Reuter"/>.

On the surface of hDim1 there are <scene name='Sandbox_502/Hdim1hyrophobic_regions/1'>two hydrophobic regions</scene>, one located towards the N-terminal region of hDim1 containing residues: Trp12, Val14, Ile18, Lue19, Phe30, Phe69, and Phe84 (pink), and one located in the clef between β3 and β4 containing residues: Met72, Met82, Met91, Ile92, Lue94, Ile102, and Trp104 (green) separated by a region of the highly conserved residues Met72, His89, and Met91 (red). The exposed hydrophobic regions coupled with the presence of five highly conserved and exposed Met residues helps solidify the role of hDim1 in protein-protein interactions with other spliceosomal proteins. In addition to the hydrophobic regions, there is also a region of conserved <scene name='Sandbox_502/Hdim1_basic_residues/1'>positively charged basic residues</scene> (Arg86, Lys88, Arg121, Arg124, Lys125, and Arg127) which may be important to binding negatively charged RNA <ref name ="Reuter"/>.

==Reduced Dominant Negative Form (hDim1-128)==

<Structure load='1pqn' size='300' frame='true' align='right' caption='Figure 2: ' scene='Sandbox_502/Hdim1-128_start_scene/2'/>

The removal of the C-terminal extension induces cell cycle arrest in [http://en.wikipedia.org/wiki/G2_phase G2], however does not affect localization, steady-state levels, or phosphorylation of the protein<ref name ="Zhang 2003"/>. This suggests that it may be the interactions to other proteins and substrates that are disrupted by the removal of the C-terminal extension <ref name ="zhang 1999"/>. It is therefore important to understand the functional changes that the presence of the C-terminal extension creates .

The structure of <scene name='Sandbox_502/Hdim1-128_start_scene/2'>hDim1-128</scene> compared to hDim1 is remarkably similar, where the same mixed β-sheet flanked by three α-helices is seen. However a prominent difference includes the loss of the β-strand comprised of residues 129-131 and 91-93. By comparing the circular dichroism spectra of hDim1 to hDim1-128 there is a decrease in α helical structure upon truncation. This suggests that the C-terminal region consists of a partially α-helical region in solution, which contradicts the findings of the hDim1 crystal structure. However, it possible that this region is naturally flexible allowing it to adopt several conformations, thereby enabling it to interact with various regions of the spliceosome as it changes through a splicing event. Interestingly, when the basic residues <scene name='Sandbox_502/Hdim1-128_arg86_and_lys88/1'>Arg86 and Lys88</scene>, which were suggested to partake in the formation of a positively charged region in hDim1 (see above), were mutated, there was a major decrease in structural stability and cooperative folding<ref name ="Zhang 2003"/>.

=Additional Resources=
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1QGV HUMAN SPLICEOSOMAL PROTEIN U5-15KD, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1PQN dominant negative human hDim1 (hDim1 1-128), in the RCSB Protein Data Bank]

=References=
<References/>

HDim1/U5-15kD

2012-08-16T04:17:53Z

2012-08-15T02:31:16Z

Kelly Hrywkiw: New page: == Structure of Prp40 == by Kelly Hrywkiw {{STRUCTURE_1o6w | PDB=1o6w | SCENE= scene name='Sandbox_504/Start_scene/1'}} __TOC__ =Introduction= Prp40 has been implicated in early...

== Structure of Prp40 ==

by Kelly Hrywkiw
{{STRUCTURE_1o6w | PDB=1o6w | SCENE= scene name='Sandbox_504/Start_scene/1'}}
__TOC__

=Introduction=

Prp40 has been implicated in early steps of [http://en.wikipedia.org/wiki/Spliceosome spliceosomal] assembly, and binding to the phosphorylated C-terminal domain (phospho-CTD) of [http://en.wikipedia.org/wiki/RNA_polymerase_II RNA polymerase II] (RNAPII)<ref name ="bonet">PMID:19722265</ref>. Two types of domains are found in Prp40, two [http://en.wikipedia.org/wiki/WW_domain WW] and six consecutive FF domains<ref name ="wiesner">PMID:12460579</ref>. WW domains contain two highly conserved tyrosine residues that bind to proline rich sequences, thereby mediating protein-protein interactions<ref name ="wiesner"/>. Both WW domains of Prp40 can interact with PPxY motifs (x is any residue) and PPѰѰP motifs (Ѱ is an aliphatic residue)<ref name ="wiesner"/>. FF domains contain two highly conserved proline residues and can be found in arrays up to six domains<ref name ="gasch">PMID:16253993</ref>. Interestingly, these tandem domains appear not to function in co-operative binding, rather they may provide multiple independent binding sites<ref name ="gasch"/>. With regards to interactions between the different domains, the WW domains bind to the splicing protein: branch point-binding protein (BBP, also known as Ms15 and ySF1) and Prp8; the FF domains interact with the splicing factor Clf1 (Syf3p in yeast), and the phospho-CTD of RNAPII<ref name ="gasch"/><ref name ="wiesner"/>.

=Role in spliceosomal assembly=

The processing of [http://en.wikipedia.org/wiki/Pre-mRNA pre-mRNA] takes place through the use of a large dynamic machine known as the spliceosome, through which [http://en.wikipedia.org/wiki/Intron introns] are removed and [http://en.wikipedia.org/wiki/Exon exons] are spliced together to create a mature [http://en.wikipedia.org/wiki/MRNA mRNA]<ref name ="architecture of"/><ref name ="structure and function">PMID:19000813</ref>. The spliceosome is comprised of five [http://en.wikipedia.org/wiki/SnRNA snRNA] molecules (snRNAs U1, U2, U4, U5, and U6) and over one hundred associated proteins<ref name ="architecture of"/><ref name ="structure and function"/>. Assembly of the spliceosome is thought to take place in a stepwise manner around the pre-mRNA transcript<ref name ="architecture of"/><ref name ="structure and function"/>. The first step involves recognition of the 5’ splice site (ss) by U1 snRNP, followed by recognition of the branch point sequence by U2 snRNP<ref name ="architecture of"/><ref name ="structure and function"/>. From this point the tri-snNRP consisting of U4/U6•U5 binds<ref name ="architecture of"/><ref name ="structure and function"/>. Together the five snRNPs form the precatalytic spliceosome which must undergo a series of changes before it can actively splice<ref name ="structural evidence">PMID:19525970</ref><ref name ="structure and function"/><ref name ="common design">PMID:19525970</ref><ref name ="architecture of">PMID:22471593</ref>.

Prp40 participates in cross-intron bridging through interactions with the 5’ss and the BBP, which brings the 5’ss and the branch point into spatial proximity<ref name ="wiesner"/>. In addition, Prp40 interacts with Prp8, such that it is possible that while one W domain interacts with Prp8 the other interacts with the 5’ss at the same time<ref name ="wiesner"/>. In fact, this interaction is thought to bridge the 5’ss to the 3’ss after U2 snRNP association displaces BBP and causes Prp8 to interact with the tri-snRNP at the 3’ss.

=Role in transcription=

RNAPII is responsible for mRNA synthesis, the first step of gene expression of eukaryotes<ref name ="cramer">PMID:11313498</ref>. During the synthesis cycle of mRNA, RNAPII interacts with up to six general [http://en.wikipedia.org/wiki/Transcription_factor transcription factors] and numerous regulatory proteins. It is comprised of 12 subunits (Rpb1 through 12) and several disordered regions including the NH2 tail of Rpb6 and Rpb12, short exposed loops in Rpb1, 2, and 8, and the CTD of the largest subunit Rpb1<ref name ="cramer"/>. The CTD is subject to hyperphosphorylation, such that RNAPII can exist in either a hyperphosphorylated form (RNAPII0) or a non-phosphorylated form (RNAPIIA) at the CTD<ref name ="phatnani">PMID:17079683</ref>. The phospho-CTD helps coordinate pre-mRNA processing events such as localizing and activating the 5’capping complex. Interestingly, when RNAPII0 is transcriptionally active in the nuclear matrix it colocalizes with splicing factors<ref name ="morris">PMID:10978320</ref>.

Prp40 can bind to the RNAPII0 at the phospho-CTD<ref name ="wiesner"/><ref name ="morris"/>. The WW domains of Prp40 have been shown to interact with the phospho-CTD; however, they do not interact with the conserved YSPTSPS sequence of phospho-CTD. The FF domains have been shown to interact with the phosphor-CTD repeats<ref name ="gasch"/>. Not all the FF domains of Prp40 exhibit the same functionality, such that the first FF domain and the fourth FF domain do not interact with the phosphor-CTD<ref name ="gasch"/>. The ability of Prp40 to bind to spliceosomal factors and the phosphor-CTD of RNAPII0 could connect the phosphor-CTD to the earliest stages of spliceosomal commitment complex formation<ref name ="morris"/>.

=Structure of Prp40=

Yeast Prp40 contains 583 residues, two WW domains, and four FF domains connected through amino acid linkers<ref name ="morris"/>.

[[Image:Prp40 domains.PNG|thumb|left|upright=4|alt=Proposed domains.|Figure1: Schematic representation of the domain organization in Prp40.]]

==The WW domains==

<Structure load='1o6w' size='250' thumb='false' align='right' caption='Figure 2: N terminal (blue) to C terminal (red) ribbon representation of the FF1 domain of Prp40' scene='Sandbox_504/Start_scene/1'/>

The overall structure of the two consecutive WW domains follows that of one triple curved antiparallel β-strand sheet connected to the other by an α-helical linker composed of residues <scene name='Sandbox_504/Linker/1'>Lys30-Glu42</scene>. The residues in each of the β strands are as follows, β1 <scene name='Sandbox_504/Wwdomain1_b1/2'>(Trp4-Asp9)</scene>, β2 <scene name='Sandbox_504/Wwdomain1_b2/1'>(Arg13-Thr19)</scene>, and β3 <scene name='Sandbox_504/Wwdomain1_b3/1'>(Lys22-Trp26)</scene> in the <scene name='Sandbox_504/Ww1/2'>first W domain</scene>, and β1 <scene name='Sandbox_504/B1_of_ww2/1'>(Trp45-Thr50)</scene>, β2 <scene name='Sandbox_504/B2_of_ww2/1'>(Lys54-Pro60)</scene>, and β3 <scene name='Sandbox_504/B3_of_ww2/1'>(Arg63-Trp67)</scene> in the <scene name='Sandbox_504/Ww2/1'>second W domain</scene>. Located on the convex surface of each of the domains lie three residues, <scene name='Sandbox_504/Hydrophobicresiduesandprotein/1'>Trp4/45, Tyr16/57, and Pro29/Leu40</scene>, that form a <scene name='Sandbox_504/Hydrophobicresiduesandprotein/2'>hydrophobic core</scene>. On the concave surface lies an aromatic pocket comprised of the residues <scene name='Sandbox_504/Aromatic_pocket_residues/1'>Tyr15/56, Tyr17/58, and Trp26/67</scene>. These pockets makes up the ligand binding sites on each of the WW domains, however they do not form one large pocket, rather <scene name='Sandbox_504/Aromatic_pocket_residues/2'>two separate small pockets </scene> that face away from each other. The linker residues Leu32 and Leu40 fold back into the hydrophobic cores of the WW domains<ref name ="wiesner"/>.

==The first FF domain (FF1)==

<Structure load='1o6w' size='250' thumb='false' align='left' caption='Figure 2: N terminal (blue) to C terminal (red) ribbon representation of the consecutive WW domains of Prp40' scene='Sandbox_504/Start_scene_ff1/1'/>

The FF1 domain of Prp40 is comprised of three alpha helices, and one 310 helix located between α2 and α3. Helices are composed of the following residues, <scene name='Sandbox_504/Ff1_a1/1'>α1</scene> (134-146), <scene name='Sandbox_504/Ff1_a2/2'>α2</scene> (154-163), <scene name='Sandbox_504/Ff1_310/1'>310</scene> (167-170), and <scene name='Sandbox_504/Ff1_a3/1'>α3</scene> (175-187). The core domain is made up of a series of aromatic and aliphatic residues. A type 1 β-turn is exhibited by the residues Asp149, Ser150, Thr151, and Trp152<ref name ="gasch"/>.

==The fourth FF domain (FF4)==

<Structure load='1o6w' size='250' thumb='false' align='right' caption='Figure 2: N terminal (blue) to C terminal (red) ribbon representation of the consecutive WW domains of Prp40' scene='Sandbox_504/Start_scene_ff4/1'/>

Domain FF4 of Prp40 exhibits compact four helical bundle fold comprised of an α1-α2-310-α3 topology. The composition of each helix is a follows <scene name='Sandbox_504/A1_of_ff4/1'>α1</scene> (Glu489-Thr507), <scene name='Sandbox_504/A2_of_ff4/1'>α2</scene> (Trp519-Leu526), <scene name='Sandbox_504/310_of_ff4/1'>310</scene> (Tyr532-Gly536) and <scene name='Sandbox_504/A3_of_ff4/1'>α3</scene> (Asp539-Phe549). There are a series of interactions between the different helices, for example Tyr532 is in contact with Phe500, Leu503, Ser523, and Arg542. A difference between FF1 and FF4 is the presence of five extra amino acids in F4 which gives the <scene name='Sandbox_504/Loop_of_ff4/1'>loop</scene> located between α 1 and α2 an extra turn. This insertion however does not increase the flexibility of F4 as compared to F1<ref name ="bonet"/>.

=Additional Resources=
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=2KFD Prp40 FF4 domain, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1O6W SOLUTION STRUCTURE OF THE PRP40 WW DOMAIN PAIR OF THE YEAST SPLICING FACTOR PRP40, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=2B7E First FF domain of Prp40 Yeast Protein, in the RCSB Protein Data Bank]

=References=
<References/>

15.5kD/Snu13/L7Ae protein

2012-08-15T02:18:41Z

Kelly Hrywkiw: New page: == Structure of the protein homologues: 15.5kD, Snu13, and L7Ae == by Kelly Hrywkiw {{STRUCTURE_1e7k | PDB=1e7k | SCENE= }} __TOC__ =Introduction= The human protein 15.5kD and ...

== Structure of the protein homologues: 15.5kD, Snu13, and L7Ae ==

by Kelly Hrywkiw
{{STRUCTURE_1e7k | PDB=1e7k | SCENE= }}
__TOC__

=Introduction=

The human protein 15.5kD and its yeast (Snu13p) and archaeal (L7Ae) homologues function in the processing of pre-ribosomal RNA as part of the box [http://en.wikipedia.org/wiki/Small_nucleolar_RNA#C.2FD_box C/D] and [http://en.wikipedia.org/wiki/Small_nucleolar_RNA#H.2FACA_box H/ACA] small ribonucleoprotein particle (sRNP – [http://en.wikipedia.org/wiki/Archaea archaea]) or small nucleolar ribonucleoprotein particle (snoRNP – [http://en.wikipedia.org/wiki/Eukarya eukarya]) nucleotide modification complexes (s(no)RNPs)<ref name ="gagnon">PMCID:PMC2802039</ref><ref name ="dobbyn">PMID:17631273</ref>. In addition, 15.5kD and Snu13p function in U4 [http://en.wikipedia.org/wiki/SnRNP small nuclear ribonucleoprotein particle] (snRNP) spliceosomal biogenesis<ref name ="dobbyn"/>. The capability to function in dual roles lies in the ability to recognize a helix-bulge-helix (kink-turn) RNA motif that is present in the different RNPs<ref name ="oruganti">PMID:15963469</ref>

A variation of the kink-turn motif, known as the kink-loop motif, can be found in the C/D and H/ACA RNAs <ref name ="gagnon"/>. Interestingly, the eukaryotic proteins and their archaeal homologue do not interact with the different motifs in the same manner, even though share a conserved sequence similarity <ref name ="oruganti"/>. For example, while L7Ae exhibits the same binding affinity for both the kink-turn and kink-loop sRNA motifs, its eukaryotic homologues only bind specifically to the kink-turn motif and discriminate against the kink-loop motif <ref name ="oruganti"/><ref name ="gagnon"/>.

Solved structures of the proteins include: 15.5kD in complex with a U4 snRNA fragment [[1E7K]], 15.5kD in complex with hPrp31 and a U4 snRNA fragment [[2OZB]], Snu13p without RNA [[1ZWZ]], [http://en.wikipedia.org/wiki/Archaeoglobus Archaeoglobus fulgidus] L7Ae-box C/D with RNA [[1RLG]], [http://en.wikipedia.org/wiki/Methanococcus_jannaschii Methanococcus jannaschii] L7Ae-H/ACA with RNA [[1RA4]], and [http://en.wikipedia.org/wiki/Pyrococcus Pyrococcus abyssi] L7Ae without RNA [[1PXW]].

=Role in pre-ribosomal RNA processing=

[http://en.wikipedia.org/wiki/Ribosome Ribosomes] consist of both RNA and protein, and are designated large ribonucleprotein (RNP) particles. Each ribosome contains two subunits (60S and 40S), four ribosomal RNAs (5S, 5.8S, 18S, and 25/28S rRNA), and approximately 75 associated proteins <ref name ="venema">PMID:10690410</ref>. The processing of the pre-rRNAs requires a complex set of posttranscriptional modification steps after [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] <ref name ="venema"/>. One such step involves extensive processing through pseudouridylation and 2’-O-ribose methylation at sites specified by various [http://en.wikipedia.org/wiki/Small_nucleolar_RNA s(no)RNAs] (C/D box s(no)RNAs specify 2’-O-ribose methylation and H/ACA s(no)RNA specify pseudouridylation) and associated proteins to form s(no)RNPs <ref name ="venema"/><ref name ="m-g">PMID:12810916</ref>. Specifically, the 5’ region of U3 s(no)RNA containing C’/D and B/C box pairs interacts with 5’-ETS and 17S/18S areas of the pre-rRNA<ref name ="m-g"/>. U3 also binds a set of proteins to form the U3 s(no)RNP complex <ref name ="gagnon"/>.

Snu13p/15.5kD/L7Ae interacts with U3 s(no)RNA through a kink-turn RNA motif <ref name ="venema"/>. The protein initiates box C/D assembly by binding the kink-turn of the C/D RNAs <ref name ="gagnon"/>. Once the s(no)RNP is fully assembled the RNA regions bind to complementary regions in target pre-rRNA. This is followed by catalysis of the methyl transferase reaction by the associated proteins <ref name ="gagnon"/>.

=Role in pre-messenger RNA splicing=

The processing of [http://en.wikipedia.org/wiki/Pre-mRNA pre-mRNA] takes place through the use of a large dynamic machine known as the spliceosome, through which [http://en.wikipedia.org/wiki/Intron introns] are removed and [http://en.wikipedia.org/wiki/Exon exons] are spliced together to create a mature [http://en.wikipedia.org/wiki/MRNA mRNA]<ref name ="architecture of"/><ref name ="structure and function">PMID:19000813</ref>. The spliceosome is comprised of five [http://en.wikipedia.org/wiki/SnRNA snRNA] molecules (snRNAs U1, U2, U4, U5, and U6) and over one hundred associated proteins<ref name ="architecture of"/><ref name ="structure and function"/>. Assembly of the spliceosome is thought to take place in a stepwise manner around the pre-mRNA transcript<ref name ="architecture of"/><ref name ="structure and function"/>. The first step involves recognition of the 5’ splice site by U1 snRNP, followed by recognition of the branch point sequence by U2 snRNP<ref name ="architecture of"/><ref name ="structure and function"/>. From this point the tri-snNRP consisting of U4/U6•U5<ref name ="architecture of"/><ref name ="structure and function"/> together with the five snRNPs form the precatalytic spliceosome, which must undergo a series of changes before it can actively splice<ref name ="structural evidence">PMID:19525970</ref><ref name ="structure and function"/><ref name ="common design">PMID:19525970</ref><ref name ="architecture of">PMID:22471593</ref>.

U4 snRNA has a 5’ stem-loop containing a kink-turn that has been shown to interact with 15.5kD <ref name ="vidovic">PMID:11163207</ref>. There is evidence to suggest that 15.5kD plays a role in late stage spliceosomal assembly, prior to splicing catalysis <ref name ="vidovic"/>. In addition, it may be involved in binding other proteins that have been found to indirectly associate with U4 snRNA such as 61k (Prp31p in yeast), as well as the 20/60/90k complex which interacts with the U4/U6 duplex <ref name ="vidovic"/>. The homologues for 60k and 90k in yeast are Prp4p and Prp3p respectively; there is no yeast homologue for 20k.

=Sructure of 15.5kD in complex with U4 snRNA fragment=

<StructureSection load='' size='500' side='left' caption='Structure of 15.5kD bound with a U4 snRNP fragment ([[1e7k]])' scene='Sandbox_503/Start_scene/2'>
__NOTOC__

=Overall structure=

15.5kD exhibits a globular domain structure, characterized by an α-β-α fold <ref name ="dobbyn"/>. The partial U4 snRNA oligonucleotide makes contacts with the protein at a pocket through nucleotide U31, which is located in the internal loop. The portion of [http://en.wikipedia.org/wiki/Oligonucleotide oligonucleotide] not contacting the protein, folds into two [http://en.wikipedia.org/wiki/Double_helix double helices] meeting to form a loop at the junction, where both are capped by a [http://en.wikipedia.org/wiki/Purine purine]. The RNA fold is stabilized through multiple hydrogen bond interactions and base stacking. The overall structure of the protein and RNA is such that there is little interaction between the RNA and protein, leaving much of the proteins surface area exposed for interactions with other components in the U4 snRNP <ref name ="vidovic"/>.

=15.5kD structure=

15.5kD is 128 amino acids long and folds into a compact α-β-α sandwich, which is the most common family of protein folds, and resembles the L30 ribosomal protein. The <scene name='Sandbox_503/B-sheet/4'>central</scene> [http://en.wikipedia.org/wiki/Beta_sheet β-sheet]contains four β-strands, one parallel and three antiparallel, and are ordered <scene name='Sandbox_503/B1/1'>β1</scene>, <scene name='Sandbox_503/B1-b4/1'>β4</scene>, <scene name='Sandbox_503/B1-b4-b2/1'>β2</scene>, and <scene name='Sandbox_503/B1-b4-b2-b3/1'>β3</scene>. To one side of the β-sheet [http://en.wikipedia.org/wiki/Alpha_helix α helices] <scene name='Sandbox_503/A1-a4-a5/1'>α1, α4, and α5</scene> are closely packed, and on the other side are <scene name='Sandbox_503/A2-a3/1'>α2 and α3</scene>. The residues 63-66 of <scene name='Sandbox_503/310_helix/3'>α3</scene> form a 310 helix that is important in RNA binding; helix α2 also contains residues important for binding<ref name ="vidovic"/>.

=U4 snRNA fragment structure=

The U4 snRNA fragment contains nucleotides <scene name='Sandbox_503/Rna_start/1'>26-47</scene> of the full length 5’stem loop. The oligonucleotide forms into two distorted A-form RNA stems (stem 1 and stem 2) that are connected by an asymmetric 5+2 internal loop (<scene name='Sandbox_503/Rna_3_nucs/1'>2 nucleotides</scene> originate from the 3’ strand and <scene name='Sandbox_503/5_strand_nucs/1'>5 nucleotides</scene> originate from the 5’ strand). The internal loop has a complex fold, where <scene name='Sandbox_503/G-a_bp_nucs/1'>four of its nucleotides</scene> from sequential G-A base pairs, and the other three are left unpaired. Of the unpaired nucleotides <scene name='Sandbox_503/U31/1'>(U31)</scene> is flipped out, and the other two <scene name='Sandbox_503/A29_a30/1'>(A30 and A29)</scene> act as purine caps by stacking onto A44 of stem two, and the G45-C28 base pair of stem 1, respectively. Between two of the G-A base pairs G32-A44 and G43-A33, the helix is overwound and causes cross stand stacking of the two adenines <scene name='Sandbox_503/A33_a44/1'>(A33 and A44)</scene>, leaving the guanines displaced. One of the unpaired adenines <scene name='Sandbox_503/A33_a44_a30/1'>(A30)</scene> also participates in the cross stacking. The overall structure represents the kink-turn motif<ref name ="vidovic"/>.

In addition to base stacking and pairing interactions that help stabilize the RNA structure, there is a network of [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] interactions that also contribute. These predominantly involve ribose 2’OH groups and nitrogen or phosphate atoms. The 2’OH of A44, A29, A33, U31, G32, and G43 hydrogen bond, or are within hydrogen bonding distance, of A30(N6), A44(N1), G45(N3), A30(P), G43(N2), and A44(P) respectively<ref name ="vidovic"/>.

=Protein-RNA interactions=

The residues of 15.5kD that play a main role in RNA binding, through interactions with the 5+2 internal loop, include those located in α2, α4, β1, and loops β1-α2, β2-α3, and α4-β4. There are four major interactions; the first involves the flipped out U31 nucleotide; the second, the sequential G-A base pairs; the third, the unpaired and stacked adenines; and the fourth, the electrostatic interactions<ref name ="vidovic"/>.

====The flipped out U31 nucleotide====

As previously mentioned the residue U31 is flipped out, which makes it an optimal site for RNA-protein interactions. It is located in pocket formed by the residues <scene name='Sandbox_503/61_65_86_100_u31_rna/1'>Glu61, Ile65, Lys86, and Ile100</scene>. Multiple hydrogen bonds and Vander walls interactions are formed between U31 and the four residues. The O4 and 3-imino group of U31 form a hydrogen bond with the main chain amide and main chain oxygen of Glu61, respectively. O4 of U31 hydrogen bonds to the amino group of Lys86, and the U31 phosphate hydrogen bonds to the main chain amide of Ile 100. In addition, the base of U31 is in Vander walls contact with the hydrophobic regions of the Ile65, Ile100, and Lys 86. U31 also forms a hydrogen bond with a residue not found in the pocket through its phosphate to the main chain oxygen of Ala39<ref name ="vidovic"/>.

====The G-A base pairs====

Of the G-A base pairs G32-A44 and G43-A33, bases G32 and G43 interact with residues Asn40, Glu41, and Lys44, found in α2 and loop β2-α1, through their exposed atoms in the major groove. Atoms within hydrogen bonding distance include N1, N2, and O6 of G32 to the carboxylate group of Glu41, N7 and O6 of G43 to the ɛ-amino group of Lys44, O6 of G32 to the main chain amide of Asn40, and N7 of G32 to the ND2 of Asn40. The adenines (A33 and A44) that base pair with the guanines (G32 and G43) do not form close interactions with the protein themselves<ref name ="vidovic"/>.

====The unpaired and stacked adenines====

The adenines that do not form base pair interactions (A29 and A30) participate in hydrophobic interactions with 15.5kD. This takes place through their side regions which are not in contact with the base pairs of stem 1 (A29) and stem two (A30). A29 packs with Arg97 in the α4β4 loop, and A30 packs with Val95 in loop α4-β4 and Lys37 on loop β1-α2<ref name ="vidovic"/>.

====The electrostatic interactions====

The RNA carries with it a negative charge due to the phosphate backbone. This charge is stabilized by several basic residues in the 15.5kD protein. The negative charges of phosphates C42 and A29 are stabilized by residues Lys44 and Agr97 respectively. Residues Arg36, Arg48, and Lys37 are in close proximity to the RNA backbone (7-8Å) and help contribute to the electrostatic state of the RNA-protein complex<ref name ="vidovic"/>.

__NOTOC__
</StructureSection>

=Structure comparison between 15.5kD, Snu13p, and L7Ae homologues=

Structurally, Snu13p and 15.5kD are more similar than either to L7Ae; however, they exhibit different binding affinities to cognate RNAs<ref name ="m-g"/><ref name ="oruganti"/>. The eukaryotic proteins exhibit very specific binding (ie. will only bind to RNA with the kink-turn motif), whereas their archaeal homologue does not (ie. will bind to RNA exhibiting either the kink-turn or kink-loop motifs)<ref name ="m-g"/><ref name ="oruganti"/>. Differences in structure between the archaeal and eukaryotic proteins lie in the α2-β2 loop and the β4-α6 loop which do not directly bind DNA, however have been shown to contribute to the structural integrity of the RNA binding elements<ref name ="oruganti"/>. In the eukaryotic proteins there is the addition of two amino acids that creates further hydrogen bonding between β2 and β4, which in turn may provide further stabilization to α2<ref name ="oruganti"/>. The N-terminus also carries an area of structural differentiation; preceding α1 in L7Ae is a random coil, whereas in 15.5kD and Snu13p there is a β-strand which may participate in further stabilization of the protein<ref name ="oruganti"/>. Overall, the structures are very similar, such that the small differences do not seem likely to contribute to their differential binding.

The structure itself may not the most important aspect when comparing the homologues, rather the amino acid composition. There are five amino acids located at the RNA binding region that are conserved within each of archaea and eukarya, however vary between the two. One such amino acid lies towards the N-terminal side of the RNA binding region, in L7Ae it is Lys26 (Methanocaldococcus jannashii), and in 15.5kD it is Gln34. Towards the C-terminal side of the RNA binding region located in loop 9 lie the four remaining residues Leu-Glu-Aal-Ala (L7Ae) and <scene name='Sandbox_503/Valserargpro/2'>Val95-Ser96-Arg97-Pro98 </scene>(15.5kD). It is the difference between these amino acids that allow L7Ae to bind the kink-loop motif<ref name ="m-g"/>.

=Additional Resources=

*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1e7k CRYSTAL STRUCTURE OF THE SPLICEOSOMAL 15.5KD PROTEIN BOUND TO A U4 SNRNA FRAGMENT, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=2ozb Structure of a human Prp31-15.5K-U4 snRNA complex, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1zwz Structural comparison of Yeast snoRNP and splicesomal protein snu13p with its homologs, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1rlg Molecular basis of Box C/D RNA-protein interaction: co-crystal structure of the Archaeal sRNP intiation complex, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1pxw Crystal structure of L7Ae sRNP core protein from Pyrococcus abyssii, in the RCSB Protein Data Bank]

=References=
<References/>

2012-07-04T20:28:45Z

Kelly Hrywkiw:

Sandbox 502

2012-07-04T20:20:25Z

Kelly Hrywkiw:

Sandbox 502

2012-07-04T18:58:56Z

Kelly Hrywkiw:

Sandbox 502

2012-07-04T18:53:14Z

Kelly Hrywkiw: New page: <!-- Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page. Or use the four-green-boxes-button to insert scrollable text adjacent to a Jmol applet. Che...

2012-06-07T17:31:24Z

2011-04-04T04:35:50Z

Kelly Hrywkiw:

== Licensing ==
{{PD-self}}