THE REFINED CRYSTAL-STRUCTURE OF THE 3C GENE-PRODUCT FROM HEPATITIS-AVIRUS - SPECIFIC PROTEINASE ACTIVITY AND RNA RECOGNITION

The virally encoded 3C proteinases of picornaviruses process the polyp rotein produced by the translation of polycistronic viral mRNA. The X- ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Cry stals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 Angstrom, b = 53.5 Angstrom c = 53.2 Angstrom, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.30. There are two molec ules of HAV 3C in the unit cell of this crystal form. The structure ha s been refined to an R factor of 0.211 (R(free) = 0.265) at 2.0-Angstr om resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two B-barrel domains. The catalytic Cys 172 S-gamma and His 44 N- epsilon 2 are separated by 3.9 Angstrom; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side cha in of Asp 84, the residue expected to form the third member of the cat alytic triad, is pointed away from the side chain of His 44 and is loc ked in an ion pair interaction with the epsilon-amino group of Lys 202 . A water molecule is hydrogen bonded to His 44 N-delta 1. The side-ch ain phenolic hydroxyl group of Tyr 143 is close to this water and to H is 44 N-delta 1 and may be negatively charged. The glutamine specifici ty for P-1 residues of substrate cleavage sites is attributed to the p resence of a highly conserved His 191 in the S-1 pocket. A very unusua l environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P-1 specifi city. HAV 3C proteinase has the conserved RNA recognition sequence KFR DI located in the interdomain connection loop on the side of the molec ule diametrically opposite the proteolytic site. This segment of polyp eptide is located between the N- and C-terminal helices, and its confo rmation results in the formation of a well-defined surface with a stro ngly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regi ons of the RNA genome during viral replication.