The role of threonine in the P2 position of bowman-birk proteinase inhibitors:: Studies on P2 variation in cyclic peptides encompassing the reactive site loop

Previously, we have described a template-assisted combinatorial peptide library based on the anti-tryptic reactive site loop of a Bowman-Birk inhibitor (BBI). Sequences that displayed inhibitory activity re-directed towards chymotrypsin were found to have a consensus binding motif, with their most striking feature being that exclusively threonine was found at the P-2 position. The present study investigates the reason for this surprising specificity by maintaining the binding motif but systematically varying the P-2 residue. From analysis of 26 variants, it is found that the requirements for inhibitory activity at P-2 are finely tuned, and in agreement with the Library work, threonine at P-2 provides optimal inhibition. In addition, peptides with threonine at P-2 are significantly less susceptible to hydrolysis. Examination of all available BBI sequences shows that threonine is very highly conserved at P-2, which implies that the functional requirement extends to the full-length BBI protein. Our results are consistent with a dual requirement for hydrophobic recognition within the S-2 pocket and maintenance of an inhibitory conformation via hydrogen bonding within the reactive-site loop. As the isolated peptide loop reproduces the active region of full-length BBI, these results explain why threonine is well conserved at P-2 in this class of inhibitor. Furthermore, they illustrate that proteinase inhibitor specificity can have characteristics that are not easily predicted from information on the substrate preferences of a proteinase. (C) 1998 Academic Press.