NS5B
From Proteopedia
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'''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. | '''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. | ||
| - | 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 | + | 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. |
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Revision as of 04:02, 21 April 2009
RNA Dependent RNA Polymerase from Hepatitis C Virus
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seen here.
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 , 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 that is proposed to move upon formation of exiting double stranded RNA.
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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.
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Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted 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 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.
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.
Image:PolyProtein.jpg
http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html Figure 4.
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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 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 . There is another site that is about 30-35Å from the active site, an , 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.
The inhibitors that bind near the 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 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.
Proteopedia Page Contributors and Editors (what is this?)
Nicolas Villanueva, Alexander Berchansky, Kody Witham, Michal Harel, David Canner
