Protein-protein recognition: An experimental and computational study of the R89K mutation in Raf and its effect on Ras binding

Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiati on. Raf/Arg89 in the center of the binding interface plays an important rol e determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vi vo. The binding to [gamma-S-35]GTP-Ras of a fusion protein between the Raf- binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mu tation, corresponding to a standard binding free energy decrease of at leas t 3.0 kcal/mol. To compute this free energy and obtain insights into the mi croscopic interactions favoring binding, we performed alchemical simulation s of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9 +/- 1.9 kcal/mol, in agreement with exp eriment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its compone nts. The binding decreases partly because of a 7 kcal/mol higher cost to de solvate Lys upon binding, compared to Arg, due to better solvent interactio ns with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to p articipate in protein-protein interfaces. Large contributions to the free e nergy change also arise from electrostatic interactions with groups up to 8 Angstrom away, namely residues 37-41 in the conserved effector domain of R as (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water mol ecules. This analysis will provide insights into the large experimental dat abase of Ras-Raf mutations.