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

Published online by Cambridge University Press:  01 January 1999

JUN ZENG
Affiliation:
Laboratoire de Biologie Structurale (C.N.R.S.), I.G.B.M.C., 1 rue Laurent Fries, 67404 Illkirch (C.U. de Strasbourg), France
MASHA FRIDMAN
Affiliation:
Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia
HIROSHI MARUTA
Affiliation:
Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia
HERBERT R. TREUTLEIN
Affiliation:
Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia
THOMAS SIMONSON
Affiliation:
Laboratoire de Biologie Structurale (C.N.R.S.), I.G.B.M.C., 1 rue Laurent Fries, 67404 Illkirch (C.U. de Strasbourg), France
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Abstract

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.

Type
Research Article
Copyright
© 1999 The Protein Society

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