2CXV

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

Summary for 2CXV

• Entry DOI: 10.2210/pdb2cxv/pdb
• Related: 1HAV 1QA7 2A4O
• Descriptor: Probable protein P3C, N-[(BENZYLOXY)CARBONYL]-L-ALANINE (3 entities in total)
• Functional Keywords: hepatitis a, picornavirus, 3c, cysteine protease, inhibitor, beta-lactone, hydrolase
• Biological source: Human hepatitis A virus Hu/Northern Africa/MBB/1978
• Cellular location: Protein VP2: Virion (By similarity). Protein VP3: Virion (By similarity). Protein VP1: Virion (By similarity). Protein VP1-2A: Virion (By similarity). Protein 2B: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Protein 2C: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Protein 3ABC: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Protein 3AB: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Protein 3A: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Protein 3B: Virion (Potential). Picornain 3C: Host cytoplasm (Potential). RNA-directed RNA polymerase 3D-POL: Host cytoplasmic vesicle membrane; Peripheral membrane protein; Cytoplasmic side (Potential): P13901
• Total number of polymer chains: 1
• Total formula weight: 24343.00

Authors

Yin, J.,Bergmann, E.M.,Cherney, M.M.,Lall, M.S.,Jain, R.P.,Vederas, J.C.,James, M.N.G. (deposition date: 2005-07-01, release date: 2005-12-27, Last modification date: 2024-10-23)

Primary citation

Yin, J.,Bergmann, E.M.,Cherney, M.M.,Lall, M.S.,Jain, R.P.,Vederas, J.C.,James, M.N.G.
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
J.MOL.BIOL., 354:854-871, 2005

PubMed 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.

PubMed: 16288920
DOI: 10.1016/j.jmb.2005.09.074

Experimental method

X-RAY DIFFRACTION (1.4 Å)