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Sandbox Reserved 1088

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Revision as of 01:15, 23 April 2015

This Sandbox is Reserved from 15/04/2015, through 15/06/2015 for use in the course "Protein structure, function and folding" taught by Taru Meri at the University of Helsinki. This reservation includes Sandbox Reserved 1081 through Sandbox Reserved 1090.

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Aedes Aegypti mosquito

Dengue Virus Non-Structural Protein NS5

The dengue virus (DENV) belongs to genus Flavivirus, which also includes the West Nile Virus, Japanese Encephalitis Virus and Yellow Fever Virus. Mosquito-borne DENV causes dengue fever which can then progress to dengue hemorrhagic fever and dengue shock syndrome. Endemic to the tropics and subtropics, it is mainly transmitted by Aedes mosquitoes. Although there are 390 million dengue infections reported annually, no specific antiviral drug or vaccine has been developed yet.

There are four serotypes of the virus (DEN-1 – DEN-4)whose respective genomes share �60% sequence identity, with �90% sequence identity within a serotype. The viral genome encodes three structural proteins (capsid, membrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). By associating with host cofactors and each other, NS proteins form multi-protein replication complexes, which comprise the viral replication machinery. The NS5 primary sequence is generally well conserved across serotypes and also shares some similarity with the NS5 protein of other flaviviruses.

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Structure

DENV NS5 (4V0Q)

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Image:DENV NS5.png

NS5 is a 104 kDa, 900 amino acid long protein, making it the largest of the DENV proteins. NS5 is also the most conserved among the viral proteins with at least 67% sequence similarity among the four serotypes. According to structural and biochemical studies, 3 functional domains exist within NS5. These being a methyltransferase (MTase) domain which spans residues 6-262 and a RNA-dependent RNA polymerase (RdRp) domain spanning residues 273-883 connected by a flexible linker domain roughly 10 amino acids (residues 263-272) in length. GREEN LINK

The RdRp is a feature unique to viruses and may be considered as a target for antiviral therapy. This domain is similar to other polymerases in that it contains finger, thumb and palm subdomains as well as making use of a common catalytic mechanism for the incorporation of nucleotides involving two metal ions (zinc in NS5) coordinated by aspartic acid residues located in the finger and thumb subdomains. Together, these subdomains and catalytic core form a tunnel through which the RNA template and nucleotides enter.

Within NS5, the MTase domain lies above the finger subdomain in such an orientation that its catalytic core (K61 D146 K180 E216) and SAH binding pockets face away from the inter-domain interface (GREEN LINK). The MTase domain is involved in 5’ capping of the newly synthesized RNA as well as methylation of certain guanine and adenine residues which contributes to evasion from host defence mechanisms. It catalyzes RNA cap methylation at both the N7 position on the cap guanosine and the 2’O on the first nucleotide of the newly synthesized (+) RNA strand. The MTase domain contains five parallel beta sheets.

The linker connects the two enzymatic domains acting like a swivel allowing the domains to adopt various relative orientations during RNA synthesis. Residues 263-266, located after the MTase C-terminus, are key in this regard. They fold into a short 310 helix (though other conformations are also thought possible) resulting in compaction of the polypeptide chain which allows for a large inter-domain interface interaction, giving the protein its globular shape. (GREEN LINK) This flexibility is a key feature as it has been demonstrated that the linker residues (268-272) N-terminal to the RdRp region enhance stability and polymerase activity and is further evidenced by results showing superior activity in the complete NS5 protein as compared to the RdRp alone, further corroborating that MTase-RdRp interactions are required to maintain RNA synthesis.

The majority of the stabilizing interactions within the inter-domain interface are polar in nature, resulting from those between charged side-chains as well as hydrogen-bonding mediated by trapped water molecules. Residues K95 and R353 form the major inter-domain polar contacts.

The inter-domain is made up of two clusters which involve residues from linker region, the RdRp finger subdomain, and the MTase domain. 1st cluster: Interaction of linker residues with both RdRp and MTase domains. Linker residue E267's side chain hydrogen bonds with with residues Y119 and R262 of the MTase domain with E269 forming a salt-bridge with RdRp domain's R361. Polar interactions between residues from the MTase domain (K95 & K96) and the RdRp domain (E296-K300) also play an important role in the cluster. 2nd cluster: Based at α5 helix residues (F348-K357)in the RdRp domain of which guanidinium of R352 interacts electrostatically with a number of residues in the MTase domain (E67, E252, Q63). A salt-bridge is also formed between K357 and D256.

Through mutation studies in DENV4, residues K95 and R353 have been implicated in critical but non-enzymatic roles in virus RNA replication and infectivity while Y119 has been shown to be necessary for MTase activity and thus a requirement for viral replication. Because of a majority of polar interactions as well as a small inter-domain buried surface area (1502A), it has been suggested that the two enzymatic domains may be able to associate and dissociate from each other with a relatively small energy penalty. Additionally, this dynamic interaction may also lend itself to making it easy to recruit other viral and/or host proteins as part of the replication complex.

1 Function
2 Disease
3 Relevance
4 Structural highlights

Function

Disease

Relevance

Structural highlights

Image:Y119-R262-E267.png

This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

↑ Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI. The global distribution and burden of dengue. Nature. 2013 Apr 25;496(7446):504-7. doi: 10.1038/nature12060. Epub 2013 Apr 7. PMID:23563266 doi:http://dx.doi.org/10.1038/nature12060
↑ Potisopon S, Priet S, Collet A, Decroly E, Canard B, Selisko B. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain. Nucleic Acids Res. 2014 Oct;42(18):11642-56. doi: 10.1093/nar/gku666. Epub 2014, Sep 10. PMID:25209234 doi:http://dx.doi.org/10.1093/nar/gku666
↑ Zhao Y, Soh TS, Zheng J, Chan KW, Phoo WW, Lee CC, Tay MY, Swaminathan K, Cornvik TC, Lim SP, Shi PY, Lescar J, Vasudevan SG, Luo D. A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication. PLoS Pathog. 2015 Mar 16;11(3):e1004682. doi: 10.1371/journal.ppat.1004682., eCollection 2015 Mar. PMID:25775415 doi:http://dx.doi.org/10.1371/journal.ppat.1004682

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Sandbox Reserved 1088

From Proteopedia

Revision as of 01:15, 23 April 2015

Dengue Virus Non-Structural Protein NS5

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@@ Line 33: / Line 33: @@
 NS5 is a 104 kDa, 900 amino acid long protein, making it the largest of the DENV proteins. NS5 is also the most conserved among the viral proteins with at least 67% sequence similarity among the four serotypes. According to structural and biochemical studies, 3 functional domains exist within NS5. These being a methyltransferase (MTase) domain which spans residues 6-262 and a RNA-dependent RNA polymerase (RdRp) domain spanning residues 273-883 connected by a flexible linker domain roughly 10 amino acids (residues 263-272) in length. GREEN LINK
-The RdRp is similar to other polymerases in that it contains finger, thumb and palm subdomains. It also contains two bound zinc atoms located specifically in the finger and thumb subdomains.
+The RdRp is a feature unique to viruses and may be considered as a target for antiviral therapy. This domain is similar to other polymerases in that it contains finger, thumb and palm subdomains as well as making use of a common catalytic mechanism for the incorporation of nucleotides involving two metal ions (zinc in NS5) coordinated by aspartic acid residues located in the finger and thumb subdomains. Together, these subdomains and catalytic core form a tunnel through which the RNA template and nucleotides enter.
 Within NS5, the MTase domain lies above the finger subdomain in such an orientation that its catalytic core (K61 D146 K180 E216) and SAH binding pockets face away from the inter-domain interface (GREEN LINK).   The MTase domain is involved in 5’ capping of the newly synthesized RNA as well as methylation of certain guanine and adenine residues which contributes to evasion from host defence mechanisms. It catalyzes RNA cap methylation at both the N7 position on the cap guanosine and the 2’O on the first nucleotide of the newly synthesized (+) RNA strand. The MTase domain contains five parallel beta sheets.