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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-β-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 ε2-nitrogen of the surface His102 by the β-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 Nε2-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 β-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. [Copyright &y& Elsevier]

Abstract: We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 Å, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the β-hairpin motif (residues 138–158), an extension of two β-strands of the C-terminal β-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sγ of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase. [Copyright &y& Elsevier]