https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Nicolas+VillanuevaProteopedia - User contributions [en]2026-10-04T01:53:55ZUser contributionsMediaWiki 1.43.8https://proteopedia.org/index.php?title=Proteopedia:Page_of_the_Year_Entrants&diff=1028315Proteopedia:Page of the Year Entrants2009-12-20T20:44:50Z<p>Nicolas Villanueva: /* Entrants */</p>
<hr />
<div>Please list your entry in [[Proteopedia:Page of the Year Competition| Proteopedia's Page of the Year Competition]] here. You can enter as many pages as you like.<br />
Add your entry to the list by adding a row in the format shown below, with a link to your user page followed by 2 hyphens and then a link to the page you're entering in the competition.<br />
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==Entrants==<br />
<br />
Entry number 1 is just an example, Eran cannot participate in the competition.<br />
<br />
# [[User:Eran Hodis]] -- [[User:Eran_Hodis/Acetylcholinesterase]]<br />
# [[User:Ramiro Barrantes]] -- [[1tdh]]<br />
# [[User:Sara Toftegaard Petersen]] [[User:Mathilde Thomsen]] [[User:Mette Trauelsen]] -- [[Nitric oxide synthase]]<br />
# [[User:Wayne Decatur]] -- [[Ribosome]] (see history for original article as of October 15, 2009)<br />
# [[User:Wayne Decatur]] -- [[User:Wayne Decatur/Plant Viral Protein p19 Suppression of RNA Silencing]]<br />
# [[User:Wayne Decatur]] -- [[User:Wayne Decatur/Suppression of RNA Silencing by Viruses]]<br />
# [[User:Wayne Decatur]] -- [[User:Wayne Decatur/Tomato aspermy virus protein 2b Suppression of RNA Silencing]]<br />
# [[User:Tzviya Zeev-Ben-Mordehai]] -- [[Intrinsically Disordered Protein]] (see history for original article as of October 19, 2009)<br />
# [[User:Tilman Schirmer]] -- [[C-di-GMP signaling]] (Note that there are several sub-pages)<br />
# [[User:Joseph Lipsick]] -- [[SRC]]<br />
# [[User:Maitreyee Mukherjee]] -- [[PHB synthase in Rhodobacter sphaeroides]]<br />
# [[User:Paula Grabowski]] -- [[Triosephosphate Isomerase]]<br />
# [[User:Céline Debarnot]] -- [[sandbox123]]<br />
# [[User:Gregg Snider]] -- [[Triose Phosphate Isomerase]]<br />
# [[User:Yash Patankar]] -- [[User:Yash Patankar/Sandbox 1]]<br />
# [[User:Julien Madouasse]] -- [[SAndbox 159]]<br />
# [[User:Mkukrishna]] -- [[User:Mkukrishna/E.COLI OMPC - CAMEL LACTOFERRIN COMPLEX]]<br />
# [[User:Lois_A._Fridmann]] -- [[HIV-1 Gag Recruitment of Tsg101 and the Viral Budding Process]]<br />
# [[User:Lois_A._Fridmann]] -- [[User:Lois A. Fridmann/Sandbox 1]]<br />
# [[User:Lois_A._Fridmann]] -- [[User:Lois A. Fridmann/Sandbox 2]]<br />
# [[User:Jianlin Cheng]] -- [[1gwp]]<br />
# [[User:Nicolas Villanueva]] -- [[NS5B]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950538NS5B2009-04-23T04:48:07Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950536NS5B2009-04-23T04:46:45Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='700' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950535NS5B2009-04-23T04:45:55Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950534NS5B2009-04-23T04:44:45Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='700' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/2'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950533NS5B2009-04-23T04:42:54Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='700' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950531NS5B2009-04-23T04:41:19Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='700' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=950468NS5B2009-04-22T20:55:09Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B.<br />
<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948938NS5B2009-04-21T15:09:36Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948937NS5B2009-04-21T15:09:20Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. IN NS5B two divalent cations coordinated by carboxyl groups (as seen in DNA polymerases) catalyze the polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo''. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948936NS5B2009-04-21T14:31:38Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948935NS5B2009-04-21T14:29:01Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate; it is not effective in each genotype of HCV, it is not well tolerated, and is expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948934NS5B2009-04-21T14:27:49Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three functions have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948933NS5B2009-04-21T14:26:24Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948932NS5B2009-04-21T14:25:53Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948931NS5B2009-04-21T14:21:30Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated ''de novo'', is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948930NS5B2009-04-21T14:20:45Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone. This viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948929NS5B2009-04-21T14:15:38Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:1849654</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948904NS5B2009-04-21T04:27:05Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
Superpositions in Figure 5 by Rould MA, and Villanueva NL<br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948903NS5B2009-04-21T04:24:08Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<ref group="xtra">PMID:17487147</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12509436</ref><references group="xtra"/><br />
<ref group="xtra">PMID:15863301</ref><references group="xtra"/><br />
[[Category: Ns5b]]<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948902NS5B2009-04-21T04:19:23Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:12589751</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
<ref group="xtra">PMID:16166071</ref><references group="xtra"/><br />
<br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948901NS5B2009-04-21T04:11:56Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
<ref group="xtra">PMID:9440688</ref><references group="xtra"/><br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948900NS5B2009-04-21T04:04:26Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
==Reference==<br />
<ref group="xtra">PMID:16824756</ref><references group="xtra"/><br />
[[Category: Hepatitis c virus]]<br />
[[Category: RNA-directed RNA polymerase]]<br />
[[Category: Hcv rna polymerase]]</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948899NS5B2009-04-21T04:02:01Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The commonly proposed explanations are that these inhibitors lock the enzyme in an inactive conformation, inhibit binding of the rGTP that binds in the area and has been shown to stimulate activity, or that this interface is critical in protein-protein interactions and disruption of these interactions by the inhibitors disrupts an oligomerization of several NS5B protomers thus making each less efficient.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
----</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948898NS5B2009-04-21T03:56:56Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from: 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5.' /><br />
'''Figure 5''' shows much of the x-ray crystallography work that has gone into NS5B to date. Each of the forty three structures included in the superposition contain at least one ligand, the ligands range from ions and small molecules to nucleotides and non-nucleoside analogue inhibitors. Positioning the structure in the familiar orientation with the <scene name='NS5B/Ns5b_with_ligands/3'>domains</scene> colored as above, it is obvious that there are two primary areas where ligands are clustering. Several nucleotides, oligonucleotides and non-nucleoside analogue inhibitors can be found in the area right around the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene>. There is another site that is about 30-35Å from the active site, an <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene>, where again ligands are clustering. There is a rGTP binding site here that seems to activate the enzyme and a nearby site where inhibitors bind and disrupt the activity of the enzyme.<br />
<br />
The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/4'>active site</scene> are thought to work by disrupting the ''primer grip'' site causing an inability of the enzyme to efficiently hold and extend a growing primer strand. The inhibitors that bind near the <scene name='NS5B/Ns5b_with_ligands/5'>allosteric site</scene> work through a poorly understood mechanism, several explanations for their ability to inhibit the enzyme have been proposed. The common propositions are that the<br />
<br />
<br />
----</div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948893NS5B2009-04-21T03:34:37Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from 1C2P, 1CSJ, 1GX5, 1GX6, 1NB4, 1NB6, 1NB7, 1HNU, 1NHV, 1OS5, 1QUV, 1YUY, 1YV2, 1YVF, 1YVX, 1YVZ, 1Z4U, 2AWZ, 2AX0, 2AX1, 2BRK, 2BRL, 2D3U, 2D3Z, 2D41, 2GIQ, 2GIR, 2HWH, 2HWI, 2I1R, 2IJN, 2JC0, 2JC1, 3BR9, 3BSA, 3BSC, 3CIZ, 3CJ0, 3CJ2, 3CJ3, 3CJ4, 3CJ5' /><br />
'''Figure 5'''<br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948892NS5B2009-04-21T03:24:36Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
----<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
'''Figure 5'''<br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948891NS5B2009-04-21T03:23:45Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''Figure 4''' is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
'''Figure 5'''<br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948890NS5B2009-04-21T03:21:55Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
Figure 4 is a depiction of each of the protein products of NS5B genomic translation. The proteins coded for by the hepatitis C virus (HCV) genome all associate with the ER membrane. The proteins are translated as one large poly-protein that is enzymatically cleaved by both host and viral proteases. Of the several proteins that are coded for by HCV, three have been identified as relevant drug targets, these are the NS3 protease and helicase domains and the NS5B RNA dependent RNA polymerase. Currently the therapy for HCV is interferon therapy often in combination with ribavirin. This therapy however is inadequate as it is not effective in each genotype of HCV, it is not well tolerated, and expensive. For these reasons many academic and industrial laboratories have been working on developing novel inhibitors of NS5B.<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948888NS5B2009-04-21T03:11:13Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948887NS5B2009-04-21T03:10:37Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
Figure 4.<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948886NS5B2009-04-21T03:09:50Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
Figure 4. [[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948884NS5B2009-04-21T03:08:25Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <br />
Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA. <br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948872NS5B2009-04-21T02:24:21Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948870NS5B2009-04-21T02:22:59Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948849NS5B2009-04-21T00:44:17Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein could move in these regions to accommodate RNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948848NS5B2009-04-21T00:39:30Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948847NS5B2009-04-21T00:33:57Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/1'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/2'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948846NS5B2009-04-21T00:32:58Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/1'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/2'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the large minor goove of dsRNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948845NS5B2009-04-21T00:32:39Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/1'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/2'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the large minor goove of dsRNA.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948844NS5B2009-04-21T00:30:36Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> P<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948843NS5B2009-04-21T00:29:02Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. The catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> P<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948842NS5B2009-04-21T00:27:21Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palms domain of NS5B and HIV reverse transcriptase in complex with DNA and an incoming dTTP then removing the HIV RT protein model while leaving the DNA where it fell into the proposed NS5B binding cleft. The catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> P<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948841NS5B2009-04-21T00:26:30Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Figure 2 is a model of NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled into the NS5B model by aligning of palms domain of NS5B and HIV reverse transcriptase in complex with DNA and an incoming dTTP then removing the HIV RT protein model while leaving the DNA where it fell into the proposed NS5B binding cleft. The catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> P<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948840NS5B2009-04-21T00:25:58Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Here is a model of NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled into the NS5B model by aligning of palms domain of NS5B and HIV reverse transcriptase in complex with DNA and an incoming dTTP then removing the HIV RT protein model while leaving the DNA where it fell into the proposed NS5B binding cleft. The catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> P<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948839NS5B2009-04-21T00:18:10Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />Here is a model of NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled into the NS5B model by aligning of palms domain of NS5B and HIV reverse transcriptase in complex with DNA and an incoming dTTP then removing the HIV RT protein model while leaving the DNA where it fell into the proposed NS5B binding cleft. The catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site.<br />
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
Each of the identified peptides that bind single stranded RNA are found in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948837NS5B2009-04-21T00:07:26Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 1. PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled in by alignment of palms domain. A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site. Catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319).<br />
The template strand is seen entering through a gap in the fingers domain, incoming nucleotide that extends the primer lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Figure 3.' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
<scene name='NS5B/Native_ns5b/3'>Domains</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
Figure 4. http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from ' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948836NS5B2009-04-21T00:02:36Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ ''in vitro'') are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='PDB IDs 2HAI (protein) 1RTD (DNA)' />NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled in by alignment of palms domain. A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site. Catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319).<br />
The template strand is seen entering through a gap in the fingers domain, incoming nucleotide that extends the primer lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Insert caption here' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
<scene name='NS5B/Native_ns5b/3'>Domains</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='NS5B' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948835NS5B2009-04-21T00:02:10Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ in vitro) are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='PDB IDs 2HAI (protein) 1RTD (DNA)' />NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled in by alignment of palms domain. A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site. Catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319).<br />
The template strand is seen entering through a gap in the fingers domain, incoming nucleotide that extends the primer lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Insert caption here' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
<scene name='NS5B/Native_ns5b/3'>Domains</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='NS5B' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanuevahttps://proteopedia.org/index.php?title=NS5B&diff=948834NS5B2009-04-21T00:00:57Z<p>Nicolas Villanueva: </p>
<hr />
<div>==RNA Dependent RNA Polymerase from Hepatitis C Virus==<br />
<br />
<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='PDB ID 2HAI' />NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, it is approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ in vitro) are the three <br />
<scene name='NS5B/Native_ns5b/4'>active site aspartates (220, 318 and 319)</scene> seen here.<br />
<br />
Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage ø 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several <br />
<scene name='NS5B/Native_ns5b/3'>domains</scene>, fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a<br />
<scene name='NS5B/Native_ns5b/6'>Beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA.<br />
<br />
<applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='PDB IDs 2HAI (protein) 1RTD (DNA)' />NS5B with B form DNA from HIV Reverse Transcriptase co-crystal model. DNA was modeled in by alignment of palms domain. A beta hairpin (residues 440-455) in the thumb domain has been moved to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site. Catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319).<br />
The template strand is seen entering through a gap in the fingers domain, incoming nucleotide that extends the primer lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole. <scene name='NS5B/Ns5b_with_dna/1'>Active site</scene> <scene name='NS5B/Ns5b_with_dna/2'>Beta-hairpin</scene> Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.Placeholder, type here.<br />
<applet load='2HAI_catalytic3.pdb' size='400' frame='true' align='right' caption='Insert caption here' /> <scene name='NS5B/Ns5b_rna_interactions/1'>NS5B RNA interactions</scene><br />
<scene name='NS5B/Native_ns5b/3'>Domains</scene><br />
<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[Image:PolyProtein.jpg]]<br />
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html<br />
== '''NS5B + LIGANDS''' ==<br />
<br />
<applet load='2HAI_ligands.pdb' size='400' frame='true' align='right' caption='NS5B' /><br />
<scene name='NS5B/Ns5b_with_ligands/3'>Domains</scene><br />
<scene name='NS5B/Ns5b_with_ligands/4'>Active site ligands</scene><br />
<scene name='NS5B/Ns5b_with_ligands/5'>Allosteric site ligands</scene></div>Nicolas Villanueva