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Double‐stranded DNA‐induced localized unfolding of HCV NS3 helicase subdomain 2
Author(s) -
Liu Dingjiang,
Windsor William T.,
Wyss Daniel F.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03280803
Subject(s) - helicase , circular dichroism , chemistry , dna , ns3 , binding site , rna helicase a , plasma protein binding , biophysics , crystallography , biochemistry , biology , rna , enzyme , protease , gene
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 (d 2Δ NS3h) by NMR and circular dichroism. Binding of dsDNA to d 2Δ NS3h induces a local unfolding of helix (α 3 ), which includes residues of conserved helicase motif VI (Q 460 RxxRxxR 467 ), and strands (β 1 and β 8 ) from the central β‐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 d 2Δ NS3h 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.