Hydrolytic Properties and Substrate Specificity of the Foot-and-Mouth Disease Leader Protease

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lbpro) between its own C-terminus and the N-terminus of VP4. Lbpro also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lbpro activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lbpro possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P7. Indeed, almost all substitutions investigated were detrimental to Lbpro activity. Analysis of structural data showed that Lbpro binds residues P1−P3 in an extended conformation, whereas residues P4−P7 are bound in a short 310 helix. The specificity of Lbpro as revealed by the substituted peptides could be explained for all positions except P5. Strikingly, Lbpro residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lbpro residues explain why Lbpro is one of the smallest, but simultaneously most specific, papain-like enzymes