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PDBsum entry 2f55
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Hydrolase/DNA
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PDB id
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2f55
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References listed in PDB file
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Key reference
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Title
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Structural and biological identification of residues on the surface of ns3 helicase required for optimal replication of the hepatitis c virus.
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Authors
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S.G.Mackintosh,
J.Z.Lu,
J.B.Jordan,
M.K.Harrison,
B.Sikora,
S.D.Sharma,
C.E.Cameron,
K.D.Raney,
J.Sakon.
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Ref.
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J Biol Chem, 2006,
281,
3528-3535.
[DOI no: ]
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PubMed id
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Abstract
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The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional
enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure
of the NS3 helicase domain (NS3h) was generated in the presence of a
single-stranded oligonucleotide long enough to accommodate binding of two
molecules of enzyme. Several amino acid residues at the interface of the two
NS3h molecules were identified that appear to mediate a protein-protein
interaction between domains 2 and 3 of adjacent molecules. Mutations were
introduced into domain 3 to disrupt the putative interface and subsequently
examined using an HCV subgenomic replicon, resulting in significant reduction in
replication capacity. The mutations in domain 3 were then examined using
recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and
RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding
assays under single turnover conditions, the mutant NS3h exhibited a similar
unwinding rate and only approximately 2-fold lower processivity than wild type
NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild
type enzyme, which was not reflective of the large reduction in HCV replicative
capacity observed in the biological experiment. Hence, the biological results
suggest that the known biochemical properties associated with the helicase
activity of NS3h do not reveal all of the likely biological roles of NS3 during
HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions
that are necessary for HCV replication.
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Figure 1.
X-ray crystal structure of NS3h bound to a 16-mer poly(dU)
substrate. Three molecules of NS3h are found in the asymmetric
unit, and two of the helicase monomers, chain A (red) and chain
B (blue), are bound to a single DNA substrate molecule (aqua).
The third molecule of the asymmetric unit (chain C, shown in
green) is bound to a separate strand of nucleic acid. The DNA
passes across the face of chain A, emerging between domains 2
and 3, where it enters the binding site of chain B.
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Figure 3.
A, the terminal region of the oligonucleotide (aqua)
interacts with NS3h chain A (red) via a hydrogen bond between
Thr^269 (T269) and the phosphate backbone and ring stacking
between nucleotide dU[1] and Trp^501 (W501; green). B, at the
point where the oligonucleotide (aqua) enters the binding groove
of chain B (blue), interactions identical to those with chain A
are observed (Trp^501-dU ring stacking and Thr^269-phosphate
hydrogen bond formation).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
3528-3535)
copyright 2006.
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