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Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA

Reprint requests to: Daniel F. Wyss, Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-L-0450, Kenilworth, NJ 07033, USA; e-mail: daniel.wyss{at}spcorp.com; fax:(908) 740-3916.

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d₂NS3h) by NMR and circular dichroism. Binding of dsDNA to d₂NS3h induces a local unfolding of helix (₃), which includes residues of conserved helicase motif VI (Q⁴⁶⁰RxxRxxR⁴⁶⁷), and strands (ß₁ and ß₈) from the central ß-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d₂NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg–Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.

Keywords: Nucleic acid-induced localized unfolding; HCV helicase; hepatitis C virus; structure–function relationships; DNA binding; helicase mechanism; NMR

Abbreviations: CSI, chemical shift index • CSP, chemical shift perturbations • ds, double-stranded • d₂NS3h, engineered version of subdomain 2 of the HCV NS3 helicase • HCV, hepatitis C virus • HSD2, dsDNA: GGCCT AAGCG-TAT-CGCTTAGGCC • HSQC, heteronuclear single quantum coherence • NA, nucleic acid • NS3h, NS3 helicase • ss, single-stranded

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				A. M. I. Lam, R. S. Rypma, and D. N. Frick Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding Nucleic Acids Res., August 2, 2004; 32(13): 4060 - 4070. [Abstract] [Full Text] [PDF]