HUMAN CYTOMEGALOVIRUS PROTEASE COMPLEXES ITS SUBSTRATE RECOGNITION SEQUENCES IN AN EXTENDED PEPTIDE CONFORMATION

Substrate hydrolysis by human cytomegalovirus (HCMV) protease is essen tial to viral capsid assembly. The interaction of HCMV protease and th e N-terminal cleavage products of the hydrolysis of R- and M-site olig opeptide substrate mimics (R and M, respectively, which span the P9-P1 positions) was studied by NMR methods. Protease-induced differential line broadening indicated that ligand binding is mediated by the P4-P1 amino acid residues of the peptides. A well-defined extended conforma tion of R from P1 through P4 when complexed to HCMV protease was evide nced by numerous transferred nuclear Overhauser effect (NOE) correlati ons for the peptide upon addition of the enzyme. NOE cross-peaks betwe en the P4 and P5 side chains placing these two groups in proximity ind icated a deviation from the extended conformation starting at P5, Simi lar studies carried out for the M peptide also indicated an extended p eptide structure very similar to that of R, although the conformation of the P5 glycine could not be established. No obvious variation in st ructure between bound R and M (notably at P4, where the tyrosine of th e R-site has been suggested to play a key role in ligand binding) coul d be discerned that might explain the observed differences in processi ng rates between R- and M-sequences. Kinetic studies, utilizing R- and M-site peptide substrates for which the P5 and P4 residues were separ ately exchanged, revealed that these positions had essentially no infl uence on the specificity constants (k(cat)/K-M). In sharp contrast, su bstitution of the P2 residue of an M-site peptide changed its specific ity constant to that of an R-site peptide substrate, and vice versa.