Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stab ilized primarily by disulfide bridges flanking the reactive-site peptide bo nd, eglin c possesses an enzyme-binding loop supported predominantly by ext ensive electrostatic/H-bonding interactions involving three Arg residues (A rg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As a n adjacent residue, the C-terminal Gly70 participates in these interactions via its cl-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H -bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the stru ctural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH2CH2CONH2) or by alpha- hydroxylacetamide (HOCH2CONH2). NMR analysis indicated that the core struct ure of eglin c was maintained in the analogues, but that the binding loop w as significantly perturbed. It was found that deletion or replacement of Gl y70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in m elting temperature. As a result, these inhibitors become substrates for the ir target enzymes. Binding assays on these analogues with a catalytically i ncompetent subtilisin BPN' mutant indicated that loss or weakening of the i nteractions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic in hibitors, the relative free energy changes (Delta Delta G) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0.999 6) with the Delta Delta G values derived from a decreased binding to the en zyme.