Binding of the protein Raf to the active form of
Ras promotes activation of the MAP kinase signaling pathway,
triggering cell growth and differentiation. Raf/Arg89 in
the center of the binding interface plays an important
role determining Ras–Raf binding affinity. We have
investigated experimentally and computationally the Raf-R89K
mutation, which abolishes signaling in vivo. The binding
to [γ-35S]GTP-Ras of a fusion
protein between the Raf-binding domain (RBD) of Raf and
GST was reduced at least 175-fold by the mutation, corresponding
to a standard binding free energy decrease of at least
3.0 kcal/mol. To compute this free energy and obtain insights
into the microscopic interactions favoring binding, we
performed alchemical simulations 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 experiment. The use of numerous runs
with three different force fields allows insights into
the sources of uncertainty in the free energy and its components.
The binding decreases partly because of a 7 kcal/mol higher
cost to desolvate Lys upon binding, compared to Arg, due
to better solvent interactions 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 participate in protein–protein interfaces.
Large contributions to the free energy change also arise
from electrostatic interactions with groups up to 8 Å
away, namely residues 37–41 in the conserved effector
domain of Ras (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 molecules.
This analysis will provide insights into the large experimental
database of Ras–Raf mutations.