Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C pro ), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C pro , determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C pro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4 -P4) and that FMDV 3C pro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C pro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C pro of an extended -ribbon that folds over the substrate binding cleft in other picornavirus 3C pro structures. Collectively, these results establish a valuable framework for the development of FMDV 3C pro inhibitors.
Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by the 3C protease at multiple, specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C protease with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C protease (FMDV 3C pro ), mutated to replace the catalytic Cys by Ala, bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5´ region of the VP1-2A cleavage junction in the viral polyprotein was determined to 2.5 Å resolution. Comparison with the free enzyme reveals significant conformational changes in 3C pro on substrate binding that lead to formation of an extended interface of contact primarily involving the P4-P2´ positions of the peptide. Strikingly, the deep S1´ specificity pocket needed to accommodate P1´-Leu only forms when the peptide binds. The substrate specificity was investigated using peptidecleavage assays to show the impact of amino acid substitutions within the P5-P4´ region of synthetic substrates. The structure of the enzyme-peptide complex explains both the marked substrate preferences for particular P4, P2 and P1 residue types as well as the relative promiscuity at P3 and on the P´ side of scissile bond. Further, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD), containing a Gln to Glu substitution, reveals an identical mode of peptide binding and explains the ability of FMDV 3C pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1´ specificity pocket and to provide direct evidence of the important contribution to proteolytic activity made by Asp 84 of the Cys-His-Asp catalytic triad. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C pro .FMDV 3C pro -peptide complex 1
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