DISSECTING THE ENERGETICS OF A PROTEIN-PROTEIN INTERACTION - THE BINDING OF OVOMUCOID 3RD-DOMAIN TO ELASTASE

An understanding of the structural basis for protein-protein interacti ons, and molecular recognition in general, requires complete character ization of binding energetics. Not only does this include quantificati on of the changes that occur in all of the thermodynamic parameters up on binding, including the enthalpy, entropy and heat capacity, but a d escription of how these changes are modulated by environmental conditi ons, most notably pH. Here, we have investigated the binding of turkey ovomucoid third domain (OMTKY3), a potent serine protease inhibitor, to the serine protease porcine pancreatic elastase (PPE) using isother mal titration calorimetry and structure-based thermodynamic calculatio ns. We find that near neutral pH the binding energetics are influenced by a shift in the pK(a) of an ionizable group, most likely histidine 57 in the protease active site. Consequently, the observed binding ene rgetics are strongly dependent upon solution conditions. Through a glo bal analysis, the intrinsic energetics of binding have been determined , as have those associated with the pK(a) shift. The protonation energ etics suggest that the drop in pK(a) is largely due to desolvation of the histidine residue. The resulting deprotonation is necessary for th e enzymatic function of elastase. Intrinsically, at 25 degrees C the b inding of OMTKY3 to PPE is characterized by an almost negligible entha lpy change, a large positive entropy change, and a large negative heat capacity change. These parameters are consistent with a model of the OMTKY3-PPE complex, which shows a large and significantly apolar prote in-protein interface. Thermodynamic calculations based upon changes th at occur in polar and apolar solvent-accessible surface area are in ve ry good agreement with the measured intrinsic binding energetics. (C) 1997 Academic Press Limited.