A Zinc Binding Site in Viral Serine Proteinases

Francesco, Raffaele De; Urbani, Andrea; Nardi, Maria Chiara; Tomei, Licia; Steinkühler, Christian; Tramontano, Anna

doi:10.1021/bi9616458

Cited by 93 publications

(73 citation statements)

References 37 publications

(43 reference statements)

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“…Disulphide bridges present in these extracellular serine proteinases are unlikely to be stable in the reducing intracellular milieu. Since NS3 is an intracellular proteinase, we proposed that the zinc-binding site is used to stabilise the relative orientation of the two b-barrel domains, thus indirectly in¯uencing the position of the catalytic triad, which is located in the crevice between the domains (De Francesco et al, 1996). However, we observe also that the histidine residue is highly conserved in all the HCV strains and HCV-related viruses.…”

Section: The Zn-binding Sitementioning

confidence: 69%

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Barbato

Cicero

Nardi

et al. 1999

Journal of Molecular Biology

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108

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The solution structure of the hepatitis C virus (BK strain) NS3 protein Nterminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zincbinding site exposed on the surface, subject to a slow conformational exchange process.

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Section: The Zn-binding Sitementioning

confidence: 69%

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Barbato

Cicero

Nardi

et al. 1999

Journal of Molecular Biology

Self Cite

108

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“…A previous study has revealed that a zinc ion is required for the structural integrity and activity of the NS3 protease (DeFrancesco et al, 1996). An electron density above the 4cr level is seen in the MIR map between the F1/A2 and D2/E2 loops.…”

Section: Overall Three-dimensional Structurementioning

confidence: 81%

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: A 2.2 Å resolution structure in a hexagonal crystal form

et al. 1998

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The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6322 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4Aprotein. There are two molecules of the NS31_180-NS4A21,-34' complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two P-barrel domains and four short a-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the &barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a &strand and an cy-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.

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“…Comparison of the NS3 protease structures in the presence and in the absence of NS4A suggested that NS4A stabilizes the N-terminal domain of the protease, thus optimizing the orientation of the catalytic triad. A characteristic feature of NS3 is the presence of a structural zinc ion that is coordinated tetrahedrally by three cysteines and a histidine residue at a site located opposite the active site (12,20,35,51). Though the NS3 protease domain is covalently attached to an RNA helicase possessing ATPase activity, the helicase domain is not required for optimal protease activity.…”

mentioning

confidence: 99%

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Trozzi

Bartholomew

Ceccacci

et al. 2003

J Virol

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115

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The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.Despite the introduction of blood-screening tests ϳ10 years ago, hepatitis C virus (HCV) is still the major cause of bloodborne chronic hepatitis, with nearly 200 million infected people worldwide. HCV infection often evolves into a chronic disease, which can lead to liver dysfunction and hepatocellular carcinoma. Current therapeutic regimens based on alpha interferon (IFN-␣) and the nucleoside analog ribavirin are only partially effective and are limited by the adverse effects of both agents (50). Given the high prevalence of this disease, developing new treatments is a major public health objective. Similarly to human immunodeficiency virus (HIV) research, most efforts to develop antiviral agents for HCV have focused on the inhibition of key viral enzymes, serine protease, helicase, and polymerase (2).The most extensively studied HCV target has been the NS3-4A serine protease, a heterodimeric enzyme comprising the N-terminal domain of the NS3 protein (amino acids 1 to 180) and the small hydrophobic NS4A protein (3). This protease cleaves the viral polyprotein at four junctions (NS3/ NS4A, NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A), and its activity is necessary for viral replication (24). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/ NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (4, 14, 28, 47). X-ray crystallography (20, 35, 51) and nuclear magnetic resonance (NMR) spectroscopy (1, 36) have shown that the NS3-4A structure is similar to that of other chymotrypsin-like serine proteases, with two domains, both composed of a ␤-barrel and two short ␣-helices. The catalytic triad comprises histidine 57, aspartate 81, and serine 139 and is located between the two domains. The central region of NS4A is an integral part of t...

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A Zinc Binding Site in Viral Serine Proteinases

Cited by 93 publications

References 37 publications

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: A 2.2 Å resolution structure in a hexagonal crystal form

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

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