Crystal Structure of an Inhibitor Complex of the 3C Proteinase from Hepatitis A Virus (HAV) and Implications for the Polyprotein Processing in HAV

Abstract

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.

Structural and Mutagenic Analysis of Foot-and-Mouth Disease Virus 3C Protease Reveals the Role of the β-Ribbon in Proteolysis

“…The distortion observed here for FMDV 3C pro is reminiscent of the lesser deformation of the catalytic triad that was observed in HAV 3C pro , the first picornaviral 3C protease for which the structure was reported (1); in that case, only the Asp residue of the triad was directed away from the active site. At the time, this finding was unprecedented, and since the deformed triad was observed in subsequent independent structure determinations (3,4), this led to the formulation of a dyad hypothesis for the catalytic mechanism for picornaviral 3C proteases which asserted that the Cys-His dyad is sufficient for activity (34). This hypothesis has only lately been challenged as a result of recent structural analyses of FMDV 3C pro (6), HAV 3C pro (37), and the 3C-like NIa protease from tobacco etch virus (TEV) (31), which observed Cys-His-Asp triads in these enzymes that closely mimicked the Ser-His-Asp triads found in serine proteases.…”

“…pro (1,3,4) and is indicative of a surprising degree of flexibility in these enzymes that has not generally been observed with chymotrypsin-like serine proteases.…”

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

“…2). Tyr 143 , which points toward the catalytic His in this enzyme was proposed to compensate for this unusual arrangement by providing sufficient electrostatic stabilization to activate the charge relay system (8,14), and, based on homology modeling, it was recently proposed that FMDV 3C pro would operate a similar system (28). However, the sequence alignment based on the structure reported here shows that neither Tyr 143 nor Lys 202 from HAV 3C pro is conserved in the FMDV enzyme (Fig.…”

Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

“…However, the hypothesis was called into question by the independent structure determinations of HAV 3C pro (4,5), in which the catalytic aspartic acid is directed away from the active site, suggesting that the Cys-His dyad is sufficient for proteolytic activity. The dispute reflects that the role of the third member of the catalytic triad has not been fully characterized.…”

“…pro , HRV 3C pro , and CVB 3C pro (14,18,20), but was not found in picornaviral proteases with Cys-His-Asp triads (4,5,7,21), suggesting that the hydrogen bond donated by residue Asn69 serves to further stabilize the conformation of the relatively longer glutamic acid side chain, which tends to be more flexible than the aspartic acid counterpart in Cys-His-Asp triads. However, the residue Asn69 is not strictly conserved in picornaviral 3C proteases even in PEV 3C pro , BEV 3C pro , and HRV 3C pro from a different HRV serotype (17) (Fig.…”