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PDBsum entry 2qeq
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References listed in PDB file
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Key reference
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Title
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Crystal structure and activity of kunjin virus ns3 helicase; protease and helicase domain assembly in the full length ns3 protein.
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Authors
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E.Mastrangelo,
M.Milani,
M.Bollati,
B.Selisko,
F.Peyrane,
V.Pandini,
G.Sorrentino,
B.Canard,
P.V.Konarev,
D.I.Svergun,
X.De lamballerie,
B.Coutard,
A.A.Khromykh,
M.Bolognesi.
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Ref.
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J Mol Biol, 2007,
372,
444-455.
[DOI no: ]
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PubMed id
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Abstract
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Flaviviral NS3 is a multifunctional protein displaying N-terminal protease
activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase
(NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to
support the separation of RNA daughter and template strands during viral
replication. In addition, NS3 assists the initiation of replication by unwinding
the RNA secondary structure in the 3' non-translated region (NTR). We report
here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase
domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant
of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of
three domains, two of which are structurally related and held to host the NTPase
and RTPase active sites. The third domain (C-terminal) is involved in RNA
binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and
in the crystals. We show that NS3:186-619 displays both ATPase and RTPase
activities, that it can unwind a double-stranded RNA substrate, being however
inactive on a double-stranded DNA substrate. Analysis of different constructs
shows that full length NS3 displays increased helicase activity, suggesting that
the protease domain plays an assisting role in the RNA unwinding process. The
structural interaction between the helicase and protease domain has been
assessed using small angle X-ray scattering on full length NS3, disclosing that
the protease and helicase domains build a rather elongated molecular assembly
differing from that observed in the NS3 protein from hepatitis C virus.
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Figure 2.
Figure 2. Structural alignment of Flavivirus helicase domain
of KUNV, DENV and YFV. The conserved motifs among superfamily 2
helicases are boxed in pink (motif I, corresponding to Walker
A), cyan (motif II, corresponding to Walker B), and gray (motif
Ia, III, IV, V and VI). The different constructs are identified
above the sequences, for KUNV (in black), and below for YFV (in
yellow), respectively. The secondary structure elements
stretches indicated refer to KUNV NS3:186–619 chain A: blue,
domain I; red, domain II; and green, domain III. In dark green
is the alignment of HCV with KUNV showing the only homologues
amino acids.
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Figure 4.
Figure 4. Superposition of the KUNV (red), DENV (blue) and
YFV (yellow) helicase structures. (a) The Walker A motif
(P-loop, in domain I), and motif V in domain II flank the ATP
binding pocket in the flaviviral helicases (stereo view). In
particular, the Walker A motif in KUNV NS3:186–619 adopts a
conformation that partially closes the ATP binding cavity,
filling the space occupied by ADP α and β-phosphate groups in
the structure of ADP-bound YFV helicase. (b) Details of the
separation between the α2 helix, in domain II, and the α9
helix, in domain III, displayed for the three flaviviral
helicase structures (color-coded as in (a)). The access site for
ssRNA in the protein central cleft is proposed to be located
between these two α-helices.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
372,
444-455)
copyright 2007.
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