Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Jen; Yang, Ueng Cheng; Hwang, Lih Hwa; Chen, Ding‐Shinn

doi:10.1128/jvi.75.17.8289-8297.2001

Cited by 69 publications

(110 citation statements)

References 50 publications

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“…The nucleophilic water (W1) loses its interaction with G417 (main-chain N) and is contacted by E291 (Oϵ1), as well as Q460 (Oϵ) in the transition state, mimicking an in-line attack to the γ-phosphorus. The importance of these motif II residues and motif VI Q460 is supported by previous mutagenesis studies (24,25,27). Our structures suggest that E291 and Q460 stabilize the positive charge developing on the attacking water, whereas the positively charged residues (K210, R464, and R467) and the metal ion likely neutralize the negative charge emerging on the γ-phosphate (Fig 2B).…”

Section: Resultssupporting

confidence: 85%

“…2). The importance of the positively charged residues is supported by previous mutagenesis and arginine methylation studies (22)(23)(24)(25). The arginine residues may play a catalytic role analogous to that of the "arginine finger" in the GTPase-activating proteins (26).…”

Section: Resultsmentioning

confidence: 83%

“…E291 coordinates the metal ion and W2 in the ground state, but makes different interactions in the transition state, as its Oϵ1 atom establishes a bifurcated hydrogen bond with W1 and W5, and Oϵ2 forms an additional interaction with H293 (Nϵ). The E291 ðOϵ2Þ-H293 (Nϵ) interaction is probably important for the substrate-stimulated (d)NTPase activity, as a H293A mutation dramatically reduces the catalytic activity associated with substrate binding (25) without impacting the basal ATPase activity (23). The nucleophilic water (W1) loses its interaction with G417 (main-chain N) and is contacted by E291 (Oϵ1), as well as Q460 (Oϵ) in the transition state, mimicking an in-line attack to the γ-phosphorus.…”

Section: Resultsmentioning

confidence: 99%

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Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

Gu¹,

Rice

2009

Proc. Natl. Acad. Sci. U.S.A.

216

348

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A virally encoded superfamily-2 (SF2) helicase (NS3h) is essential for the replication of hepatitis C virus, a leading cause of liver disease worldwide. Efforts to elucidate the function of NS3h and to develop inhibitors against it, however, have been hampered by limited understanding of its molecular mechanism. Here we show x-ray crystal structures for a set of NS3h complexes, including ground-state and transition-state ternary complexes captured with ATP mimics (ADP · BeF 3 and ADP · AlF − 4 ). These structures provide, for the first time, three conformational snapshots demonstrating the molecular basis of action for a SF2 helicase. Upon nucleotide binding, overall domain rotation along with structural transitions in motif V and the bound DNA leads to the release of one base from the substrate base-stacking row and the loss of several interactions between NS3h and the 3′ DNA segment. As nucleotide hydrolysis proceeds into the transition state, stretching of a "spring" helix and another overall conformational change couples rearrangement of the (d)NTPase active site to additional hydrogen-bonding between NS3h and DNA. Together with biochemistry, these results demonstrate a "ratchet" mechanism involved in the unidirectional translocation and define the step size of NS3h as one base per nucleotide hydrolysis cycle. These findings suggest feasible strategies for developing specific inhibitors to block the action of this attractive, yet largely unexplored drug target.antivirals | motor protein | step size | superfamily 2 | transition state H epatitis C virus (HCV) is a 9.6 kb positive-sense, singlestranded RNA (ssRNA) virus of the Flaviviridae family. It is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). A protective vaccine is not available and the existing treatment is frequently ineffective. Like other positive-strand RNA viruses, HCV replicates in close association with modified intracellular membranes (2), where the viral replicase complex catalyzes accumulation of progeny RNA genomes through a negative-strand intermediate. The helicase domain of viral nonstructural protein 3 (NS3h) is an enzymatic component of this replication apparatus and is essential for HCV propagation; the precise role of the helicase, however, remains obscure [reviewed in (2, 3)].NS3h is classified as a superfamily-2 (SF2) DExH helicase. It has polynucleotide-stimulated (d)NTPase activity (4) and can unwind both RNA and DNA in a 3′-5′ direction (5, 6). Structures of NS3h in complex with single-stranded DNA (ssDNA) have shown that DNA binds in a groove between domain 3 and the RecA-like domains, with the bases stacked in a row between two "bookend" residues (V432 in the domain 2 β-hairpin and W501 in domain 3) (7,8). The structure of the substrate DNA remains ambiguous, however, as the pucker conformations assigned to the sugar groups differ between reports. Whereas ATP binding has been suggested between NS3h domains 1 and 2 (7), the atomic details of nucleotide binding and hydrolysis have...

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

Gu¹,

Rice

2009

Proc. Natl. Acad. Sci. U.S.A.

216

348

View full text Add to dashboard Cite

show abstract

“…It was therefore somewhat surprising when a co-structure of a short oligonucleotide bound to HCV helicase showed the DNA bound to the cleft separating domain 3 from the RecA-like domains (Fig (2a)) [113]. Extensive sitedirected mutagenesis of HCV helicase has since confirmed the validity of the HCV helicase-DNA structure and demonstrated that the observed protein-nucleic acid contacts are needed for efficient unwinding [131][132][133][134][135][136]. Furthermore, a similar DNA binding cleft, which is perpendicular to the ATP binding cleft, has since been seen in co-structures of two other non-ring SF1 helicases with DNA [137,138].…”

Section: Mechanism Of Action Of Non-ring Helicases (Sf1 Sf2 Helicases)mentioning

confidence: 99%

“…The importance of several residues that contact the backbone in the HCV-DNA structure has been verified using site-directed mutagenesis. Critical nucleic acid back-bone-binding HCV residues include Thr269 [131], Thr411 [131], Thr450 [74], Arg461 [132] and Arg393 [136]. Only one HCV helicase amino acid side chain, that of Trp501, is known to contact the bases [113].…”

Section: Mechanism Of Action Of Non-ring Helicases (Sf1 Sf2 Helicases)mentioning

confidence: 99%

Understanding Helicases as a Means of Virus Control

Frick¹,

Lam²

2006

CPD

103

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Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

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Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model

et al. 2007

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Hepatitis C virus NS3 helicase is an enzyme that unwinds double-stranded polynucleotides in an ATP-dependent reaction. It provides a promising target for small molecule therapeutic agents against hepatitis C. Design of such drugs requires a thorough understanding of the dynamical nature of the mechanochemical functioning of the helicase. Despite recent progress, the detailed mechanism of the coupling between ATPase activity and helicase activity remains unclear. Based on an elastic network model (ENM), we apply two computational analysis tools to probe the dynamical mechanism underlying the allosteric coupling between ATP binding and polynucleotide binding in this enzyme. The correlation analysis identifies a network of hot-spot residues that dynamically couple the ATP-binding site and the polynucleotide-binding site. Several of these key residues have been found by mutational experiments as functionally important, while our analysis also reveals previously unexplored hot-spot residues that are potential targets for future mutational studies. The conformational changes between different crystal structures of NS3 helicase are found to be dominated by the lowest frequency mode solved from the ENM. This mode corresponds to a hinge motion of the highly flexible domain 2. This motion simultaneously modulates the opening/closing of the domains 1-2 cleft where ATP binds, and the domains 2-3 cleft where the polynucleotide binds. Additionally, a small twisting motion of domain 1, observed in both mode 1 and the computed ATP binding induced conformational change, fine-tunes the binding affinity of the domains 1-3 interface for the polynucleotide. The combination of these motions facilitates the translocation of a single-stranded polynucleotide in an inchworm-like manner.

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Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Cited by 69 publications

References 50 publications

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

Understanding Helicases as a Means of Virus Control

Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model

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