2a4o | pdb_00002a4o
Dual modes of modification of Hepatitis A virus 3C protease by a serine derived beta-lactone: selective crytstallization and high resolution structure of the His102 adduct
Structural highlights
FunctionPOLG_HAVMB Capsid proteins VP1, VP2, and VP3 form a closed capsid enclosing the viral positive strand RNA genome. All these proteins contain a beta-sheet structure called beta-barrel jelly roll. Together they form an icosahedral capsid (T=3) composed of 60 copies of each VP1, VP2, and VP3, with a diameter of approximately 300 Angstroms. VP1 is situated at the 12 fivefold axes, whereas VP2 and VP3 are located at the quasi-sixfold axes. The capsid interacts with HAVCR1 to provide virion attachment to target cell (By similarity). Protein VP0: VP0 precursor is a component of immature procapsids. The N-terminal domain of VP0, protein VP4, is needed for the assembly of 12 pentamers into the icosahedral structure. Unlike other picornaviruses, HAV VP4 does not seem to be myristoylated and has not been detected in mature virions, supposedly owing to its small size (By similarity). VP1-2A precursor is a component of immature procapsids and corresponds to an extended form of the structural protein VP1. The C-terminal domain of VP1-2A, protein 2A, acts as an assembly signal that allows multimerization of VP1-2A and formation of pentamers of VP1-VP2-VP3 trimers. It is proteolytically removed from the precursor by a host protease and does not seem to be found in mature particles (By similarity). Protein 2B and 2BC precursor affect membrane integrity and cause an increase in membrane permeability (By similarity). Protein 2C: Associates with and induces structural rearrangements of intracellular membranes. It displays RNA-binding, nucleotide binding and NTPase activities (By similarity). Protein 3A, via its hydrophobic domain, serves as membrane anchor to the 3AB and 3ABC precursors (By similarity). The 3AB precursor interacts with the 3CD precursor and with RNA structures found at both the 5'- and 3'-termini of the viral genome. Since the 3AB precursor contains the hydrophobic domain 3A, it probably anchors the whole viral replicase complex to intracellular membranes on which viral RNA synthesis occurs (By similarity). The 3ABC precursor is targeted to the mitochondrial membrane where protease 3C activity cleaves and inhibits the host antiviral protein MAVS, thereby disrupting activation of IRF3 through the IFIH1/MDA5 pathway. In vivo, the protease activity of 3ABC precursor is more efficient in cleaving the 2BC precursor than that of protein 3C. The 3ABC precursor may therefore play a role in the proteolytic processing of the polyprotein (By similarity). Protein 3B is covalently linked to the 5'-end of both the positive-strand and negative-strand genomic RNAs. It acts as a genome-linked replication primer (By similarity). Protease 3C: cysteine protease that generates mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, it binds to viral RNA, and thus influences viral genome replication. RNA and substrate bind cooperatively to the protease. Also cleaves host proteins such as PCBP2 (By similarity). RNA-directed RNA polymerase 3D-POL replicates genomic and antigenomic RNA by recognizing replications specific signals (By similarity). Publication Abstract from PubMedHepatitis 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. 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[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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