ELECTROSTATIC CONTRIBUTIONS TO PROTEIN-PROTEIN BINDING AFFINITIES - APPLICATION TO RAP RAF INTERACTION/

The challenge of evaluating absolute binding free energies of protein- protein complexes is addressed using the scaled Protein Dipoles Langev in Dipoles (PDLD/S) model in combination with the Linear Response Appr oximation (LRA). This is done by taking the complex between Rap1A (Rap ) and the p21(ras) binding domain of c-Raf (Raf-RBD) (Nassar et al., N ature 375:554-560, 1995) as a model system. Several formulations and d ifferent thermodynamic cycles are explored taking advantage of the LRA method and considering the protein reorganization during complex form ation. The performance of different approximations is examined by comp aring the calculated and observed absolute binding energies for the na tive complex and some of its mutants. The evaluation of the contributi ons of individual residues to the binding free energy, which is referr ed to here as group contributions is also examined. Special attention is paid to the role of the ''dielectric constant,'' epsilon(in) which is in fact a scaling factor that represents the contributions that are treated implicitly. It is found that explicit consideration of protei n relaxation is crucial for obtaining reasonable results with small va lues of epsilon(in) but it is also found that such a treatment of prot ein-protein interactions is very challenging and does not always give stable results. This indicates that more advanced explicit calculation s should be based on experimentally determined structures of both the complex and the isolated proteins. Nevertheless, it is demonstrated th at the qualitative trend of the effect of mutations can be reproduced by considering the effect of protein reorganization implicitly, using epsilon(in) similar to 25 for ionized residues and epsilon(in) similar to 4 for polar residues. Thus, it is concluded that an explicit treat ment of solvent relaxation (which is common to current continuum model s) does not provide sufficient compensation for turning off the charge s of ionized residues on the interaction surface of the Raf-RBD/Rap co mplex. Representing the missing contribution by large epsilon(in) can, of course, reproduce the observed effect of ionized residues, but now the contribution of uncharged residues will be largely underestimated . Regardless of these conceptual problems, it is established that a ve ry simple nonrelaxed approach, where the relaxation of both the protei n and the solvent are considered implicitly, can provide an effective qualitative way for evaluating group contributions, using large and sm all values for epsilon(in) of ionized and neutral residues, respective ly. As much as the actual system studied is concerned we find that mor e residues than generally assumed play a role in Raf-RBD/Rap interacti on. This includes residues that are not located at the protein-protein interaction surface. These residues contribute to the binding energy through direct charge-charge interaction without leading to drastic st ructural changes. The overall contribution of the surface residues is quite significant since Raf and Rap are positively and negatively char ged, respectively, and their charges are distributed along the interac tion site between the two proteins. (C) 1998 Wiley-Liss, Inc.