Crystal Structure and Activity of Kunjin Virus NS3 Helicase; Protease and Helicase Domain Assembly in the Full Length NS3 Protein

Abstract

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-transferase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication.We solved the three-dimensional structure (at 3.1 Å resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus (1). We showed that NS3:186-619 displays both ATPase and RTPase activity, 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. S.A.X.S. data were collected on the X33 camera of the EMBL on the storage ring DORIS III (DESY, Hamburg, Germany). The data were recorded using a MAR345 two-dimensional image plate detector at a sample-detector distance of 2.7 m and a wavelength of λ = 1.5 Å, covering the range of momentum transfer 0.012 < s < 0.45 Å -1 (s = 4π sinθ/λ, where 2θ is the scattering angle). The solutions containing NS3FL at 3.0, 1.5 and 0.75 mg/ml in Hepes 10 mM, NaCl 300 mM and protease inhibitors cocktail (Roche), with the addition of 2 mM DTT to reduce radiation damage, were placed in a 70 µl cell with polystyrene windows and exposed to X-ray beam. To check for radiation damage, the data were collected in two successive 2-minute exposures, and no changes were observed in the scattering patterns with time, i.e., there was no measurable radiation damage. All measurements were performed at 15°C. The data were averaged after normalization to the intensity of the incident beam and the scattering of the buffer was subtracted. The difference curves were scaled for the solute concentration and extrapolated to infinite dilution. All data manipulations were performed using the program package PRIMUS (2).The forward scattering I(0) and the radius of gyration (R g ) were evaluated using the Guinier approximation (3); the maximum diameter D max of the particle were computed from the entire scattering patterns using program GNOM (4), and the excluded volume V p of the particle was computed from the Porod invariant (5). The molecular masses of the solutes were evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (66 kDa). The ab initio shape reconstruction was performed using the program GASBOR (6), using the total number of the residues in NS3FL (619).The estimated molecular mass of the NS3FL sample, 65±5 kDa, is compatible with mon...

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

“…This interdomain flexibility may have mechanistic implications for flaviviral NS3 proteins. Small angle X-ray scattering (SAXS) data from full-length Dengue and Kunjin NS2B/NS3 proteins indicate an elongated shape and support the domain assembly observed in the flaviviral crystal structures (19,23). Although there are significant differences between hapacivirus and flavivirus NS3 proteins, including the different cofactor, these results reinforced our interest to investigate if the domain orientation observed previously (13) is representative of the HCV NS3/4A domain organization in solution.…”

Flaviviral helicase: Insights into the mechanism of action of a motor protein

“…Briefly, the covariance matrix of protein coordinates during the simulation can be diagonalized, with the highest eigenvalues representing the larger positional fluctuations, and the corresponding eigenvectors representing the main collective motions within the protein. In our simulation, the covariance matrix of Cas in the absence of ATP shows a large anticorrelated motion involving domain II (amino acids 160-240) and III (360-451) indicating a scissor like swinging of the two domains, as already observed in normal mode analysis [3]. On the contrary, such low frequency structural rearrangement almost vanishes in the presence of ATP, as demonstrated also by the difference between the sum of the two principal covariance eigenvalues in the presence and in the absence of ATP, varying from 3.1 to 5.1 (arbitrary units), respectively.…”

“…The overall F-Hel structure resembles that of an equilateral triangle, where each domain is placed at a vertex. The center of the triangle, where the three domains converge, contains an evident cleft that was first hypothesized [3], and then observed, to host ssRNA during protein activity [4]. The ssRNA access site in the cleft is located between a-helix 2 of domain II, the b-hairpin protruding from domain II toward domain III, and the C-terminal a-helix 6 of domain III; being surrounded by two a-helices, we will refer to the ssRNA access site as the ''a-helical gate''.…”

“…Different mechanistic models describe the unwinding process as ''active'' (the protein induces double strand melting) or ''passive'' (the protein takes advantage of thermal fluctuation causing ds opening and progresses along ssRNA) [5]. For F-Hel, we previously proposed that part of the protein mechanical action on dsRNA could result from a ''scissor-like'' structural rearrangement, involving mainly domains II and III, occurring near the ssRNA access into the protein cleft [3]. Such conformational rearrangement of F-Hel, suggested by normal mode analysis, essentially describes ''open'' and ''closed'' states of the a-helical gate.…”

Development of a new microplate for micro-scale vapour diffusion

“…Normal mode analysis of the NS3:186-619 helicase construct indicates the presence of a scissors-like oscillation involving domains II and III, resulting in opening/closure of the ssRNA binding cleft entrance. Such intramolecular scissors-movements may be part of the inchworm mechanism by providing a strain component for dsRNA unwinding [1]. NS3:186-619 displays both ATPase and RTPase activity and can unwind a dsRNA substrate.…”