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Article: Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications

TitleFlexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications
Authors
Issue Date2010
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 2010, v. 285 n. 24, p. 18817-18827 How to Cite?
AbstractThe dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B 18NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn 2+ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu 173 and Pro 174 or replacing Pro 174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro 176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/171780
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLuo, Den_US
dc.contributor.authorWei, Nen_US
dc.contributor.authorDoan, DNen_US
dc.contributor.authorParadkar, PNen_US
dc.contributor.authorChong, Yen_US
dc.contributor.authorDavidson, ADen_US
dc.contributor.authorKotaka, Men_US
dc.contributor.authorLescar, Jen_US
dc.contributor.authorVasudevan, SGen_US
dc.date.accessioned2012-10-30T06:17:03Z-
dc.date.available2012-10-30T06:17:03Z-
dc.date.issued2010en_US
dc.identifier.citationJournal Of Biological Chemistry, 2010, v. 285 n. 24, p. 18817-18827en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/171780-
dc.description.abstractThe dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180-618) by an 11-amino acid linker (169-179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B 18NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn 2+ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu 173 and Pro 174 or replacing Pro 174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro 176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshAdenosine Diphosphate - Chemistryen_US
dc.subject.meshCloning, Molecularen_US
dc.subject.meshCrystallography, X-Ray - Methodsen_US
dc.subject.meshDna Helicases - Chemistryen_US
dc.subject.meshGlycine - Chemistryen_US
dc.subject.meshManganese - Chemistryen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshMutagenesis, Site-Directeden_US
dc.subject.meshMutationen_US
dc.subject.meshPeptide Hydrolases - Chemistryen_US
dc.subject.meshProtein Conformationen_US
dc.subject.meshProtein Structure, Secondaryen_US
dc.subject.meshProtein Structure, Tertiaryen_US
dc.subject.meshRna - Chemistryen_US
dc.subject.meshRna Helicases - Metabolismen_US
dc.subject.meshSerine Endopeptidases - Metabolismen_US
dc.subject.meshViral Nonstructural Proteins - Metabolismen_US
dc.titleFlexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implicationsen_US
dc.typeArticleen_US
dc.identifier.emailKotaka, M:masayo@hku.hken_US
dc.identifier.authorityKotaka, M=rp00293en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1074/jbc.M109.090936en_US
dc.identifier.pmid20375022-
dc.identifier.scopuseid_2-s2.0-77953306294en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-77953306294&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume285en_US
dc.identifier.issue24en_US
dc.identifier.spage18817en_US
dc.identifier.epage18827en_US
dc.identifier.isiWOS:000278453900072-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLuo, D=14020135800en_US
dc.identifier.scopusauthoridWei, N=36464740500en_US
dc.identifier.scopusauthoridDoan, DN=36463328500en_US
dc.identifier.scopusauthoridParadkar, PN=6508270871en_US
dc.identifier.scopusauthoridChong, Y=36463402900en_US
dc.identifier.scopusauthoridDavidson, AD=7402001807en_US
dc.identifier.scopusauthoridKotaka, M=6604073578en_US
dc.identifier.scopusauthoridLescar, J=6603844493en_US
dc.identifier.scopusauthoridVasudevan, SG=35498684400en_US

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