title: Unmasking the active helicase conformation of nonstructural protein 3 from hepatitis C virus.
abstract: The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific β-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the β-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3ΔC7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3ΔC7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3ΔC7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3ΔC7 is independent of the substrate 3'-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Steve C
authors.initials: SC
authors.lastname: Ding
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Kohlway
authors.forename: Anna M
authors.initials: AM
authors.lastname: Pyle
mesh_terms: D016174:Hepacivirus / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D012313:RNA; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases