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spinqualitylabelsall models 2cxv, resolution 1.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Dual Modes of Modification of Hepatitis A Virus 3C Protease by a Serine-Derived betaLactone: Selective Crystallization and High-resolution Structure of the His-102 Adduct

Overview

Hepatitis A virus (HAV) 3C proteinase is a member of the picornain, cysteine proteases responsible for the processing of the viral, polyprotein, a function essential for viral maturation and infectivity., This and its structural similarity to other 3C and 3C-like proteases make, it an attractive target for the development of antiviral drugs. Previous, solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C, protein, which displays catalytic properties indistinguishable from the, native enzyme, is irreversibly inactivated by, N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the, sulfur atom at the active site Cys172. However, crystallization of an, enzyme-inhibitor adduct from the reaction mixture followed by X-ray, structural analysis shows only covalent modification of the, epsilon2-nitrogen of the surface His102 by the beta-lactone with no, reaction at Cys172. Re-examination of the heteronuclear multiple quantum, coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates, that dual modes of single covalent modification occur with a >/=3:1 ratio, of S-alkylation of Cys172 to N-alkylation of His102. The latter product, crystallizes readily, probably due to the interaction between the phenyl, ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of, a neighboring protein molecule in the crystal. Furthermore, significant, structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84, accepting one hydrogen bond from His44. Although the 3C protease modified, at Cys172 is catalytically inactive, the singly modified His102, N(epsilon2)-alkylated protein displays a significant level of enzymatic, activity, which can be further modified/inhibited by, N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal), or excessive amount of the same beta-lactone inhibitor (in solution). The, success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness, of this new crystal form in the study of enzyme-inhibitor interactions in, the proteolytic active site.

About this Structure

2CXV is a Single protein structure of sequence from Hepatitis a virus with BBL as ligand. Active as Picornain 3C, with EC number 3.4.22.28 Full crystallographic information is available from OCA.

Reference

Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site., Yin J, Bergmann EM, Cherney MM, Lall MS, Jain RP, Vederas JC, James MN, J Mol Biol. 2005 Dec 9;354(4):854-71. Epub 2005 Oct 14. PMID:16288920

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