Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action. J Biol Chem 2003 Nov 07;278(45):44514-24
Date
08/29/2003Pubmed ID
12944414Pubmed Central ID
PMC3571693DOI
10.1074/jbc.M306444200Scopus ID
2-s2.0-0242582359 (requires institutional sign-in at Scopus site) 57 CitationsAbstract
The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The roles of two such motifs are examined here. The first motif (YRGXDV) forms a loop that connects SF2 helicase motifs 4 and 5, at the tip of which is Arg393. When Arg393 is changed to Ala, the resulting protein (R393A) retains a nucleic acid stimulated ATPase but cannot unwind RNA. R393A also unwinds DNA more slowly than wild type and translocates poorly on single-stranded DNA (ssDNA). DNA and RNA stimulate ATP hydrolysis catalyzed by R393A like the wild type, but the mutant protein binds ssDNA more weakly both in the presence and absence of the non-hydrolyzable ATP analog ADP(BeF3). The second motif (DFSLDPTF) forms a loop that connects two anti-parallel sheets between SF2 motifs 5 and 6. When Phe444 in this Phe-loop is changed to Ala, the resulting protein (F444A) is devoid of all activities. When Phe438 is changed to Ala, the protein (F438A) retains nucleic acid-stimulated ATPase, but does not unwind RNA. F438A unwinds DNA and translocates on ssDNA at about half the rate of the wild type. Equilibrium binding data reveal that this uncoupling of ATP hydrolysis and unwinding is due to the fact that the F438A mutant does not release ssDNA upon ATP binding like the wild type. A model is presented explaining the roles of the Arg-clamp and the Phe-loop in the unwinding reaction.
Author List
Lam AM, Keeney D, Frick DNAuthor
David N. Frick PhD Associate Professor in the Chimistry & Biochemistry department at University of Wisconsin - MilwaukeeMESH terms used to index this publication - Major topics in bold
Adenosine TriphosphateAmino Acid Sequence
Animals
Binding Sites
Conserved Sequence
DEAD-box RNA Helicases
DNA
DNA, Single-Stranded
Hepacivirus
Humans
Hydrolysis
Models, Molecular
Molecular Sequence Data
Molecular Structure
Mutagenesis, Site-Directed
Nucleoside-Triphosphatase
Protein Conformation
RNA
RNA Helicases
Sequence Alignment
Serine Endopeptidases
Structure-Activity Relationship
Substrate Specificity
Viral Nonstructural Proteins
