title: 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.
abstract: 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.
authors.affiliation: CIHR Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Jiang
authors.initials: J
authors.lastname: Yin
authors.forename: Ernst M
authors.initials: EM
authors.lastname: Bergmann
authors.forename: Maia M
authors.initials: MM
authors.lastname: Cherney
authors.forename: Manjinder S
authors.initials: MS
authors.lastname: Lall
authors.forename: Rajendra P
authors.initials: RP
authors.lastname: Jain
authors.forename: John C
authors.initials: JC
authors.lastname: Vederas
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
mesh_terms: D000086042:3C Viral Proteases; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D030041:Hepatitis A virus / Q000201:enzymology*; D007783:Lactones / Q000737:chemistry* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D015202:Protein Engineering; D012694:Serine; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D007783:Lactones; D014764:Viral Proteins; D012694:Serine; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases