Interscaffolding additivity: binding of P1 variants of bovine pancreatic trypsin inhibitor to four serine proteases

Abstract

Different families of protein inhibitors of serine proteases share similar conformation of the enzyme-binding loop, while their scaffoldings are completely different. In the enzyme-inhibitor complex, the P1 position of the loop makes numerous contacts within the S1 pocket and significantly influences the energy of the interaction. Here, we determine the association energies (ΔGa values) for the interaction of coded P1 variants of bovine pancreatic trypsin inhibitor (BPTI) with bovine β-trypsin (BT), anionic salmon trypsin (AST), bovine α-chymotrypsin (BCHYM), and human neutrophil elastase (HNE). The respective ΔGa ranges are 15, 13, 9, and 8 kcal mol−1(1 cal = 4.18 J). Next, through interscaffolding additivity cycles, we compare our set of ΔGa values determined for BCHYM and HNE with similar data sets available in the literature for three other inhibitor families. The analysis of the cycles shows that 27 to 83 % of cycles fulfil the criteria of additvity. In one particular case (comparison of associations of P1 variants of BPTI and OMTKY3 with BCHYM) there is a structural basis for strongly non-additive behaviour. We argue that the interscaffolding additvity depends on sequential and conformational similarities of sites where the mutation(s) are introduced and on the particular substitution. In the second interscaffolding analysis, we compare binding of the same P1 mutants to BT and AST. The high correlation coefficient shows that both trypsins recognize with comparable strength the non-cognate side-chains. However, the cognate Arg and Lys side-chains are recognized significantly more strongly by AST.