ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS AND THE EFFECTS OF AMINO-ACID MUTATIONS ON THEIR ENERGETICS - THE IMPORTANCE OF WATER-MOLECULES INTHE BINDING EPITOPE

A modelling analysis has been conducted to assess the determinants of binding strength and specificity for three crystal complexes; the anti -hen egg white lysozyme antibody D1.3 complexed with hen egg white lys ozyme (HEL), the D1.3 antibody complexed with the anti-lysozyme antibo dy E5.2, and barnase complexed with barstar. The strengths of individu al binding components within these interfaces are evaluated using a mo del of binding free energy that is based on pairwise surface preferenc es. Ln all cases the energetics of binding are dominated by a relative ly small number of interfacial residues that define the binding epitop e. A precise geometric arrangement of these residues was not found; th ey were either localized to one region, or distributed throughout the binding interface. Surprisingly, interfacial crystal water molecules w ere calculated to contribute around 25% of the total calculated bindin g strength. Theoretical alanine mutations were completed by atomic del etions of the wild-type complexes. Strong correlations were observed b etween the calculated changes in binding free energy (Delta Delta G(ca lculated)) and the experimental values (Delta Delta G(observed)) for a ll but three of the 30 single residue mutations in the D1.3-HEL, D1.3- E5.2 and barnase-barstar systems and for all of the double mutations i n the barnase-barstar system. This analysis finds that the observed di fferences in binding strength are consistent with a model that account s for the changes in binding energy from the direct contacts between e ach member of the complex and indirect changes due to released crystal lographic water molecules that are near the mutation site. The observe d energy changes for double mutations in the barnase-barstar system is fully accounted for by considering water molecules bound jointly by e ach member of the complex. (C) 1997 Academic Press Limited.