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COMBINE Analysis of the Specificity of Binding of Ras Proteins to their Effectors

The small guanosine triphosphate (GTP)-binding proteins of the Ras family are involved in many cellular pathways leading to cell growth, differentiation, and apoptosis. Understanding the interaction ofRaswith other proteins is of importance not only for studying signalling mechanisms but also, because of their medical relevance as targets, for anticancer therapy. To study their selectivity and specificity, which are essential to their signal transfer function, we performed COMparative BINding Energy (COMBINE) analysis for 122 differentwild- type andmutant complexes between the Ras proteins, Ras and Rap, and their effectors, Raf and RalGDS. The COMBINE models highlighted the amino acid residues responsible for subtle differences in binding of the same effector to the two different Ras proteins, aswell asmore significant differences in the binding of the two different effectors (RalGDS and Raf) to Ras. The study revealed that E37, D38, and D57 in Ras are nonspecific hot spots at its effector interface, important for stabilization of both the RalGDS– Ras and Raf– Ras complexes. The electrostatic interaction between a GTP analogue and the effector, either Raf or RalGDS, also stabilizes these complexes. The Raf– Ras complexes are specifically stabilized by S39, Y40, and D54, and RalGDS– Ras complexes by E31 and D33. Binding of a small molecule in the vicinity of one of these groups of amino acid residues could increase discrimination between the Raf– Ras and RalGDS– Ras complexes. Despite the different size of the RalGDS– Ras and Raf– Ras complexes, we succeeded in building COMBINE models for one type of complex that were also predictive for the other type of protein complex. Further, using system-specific models trained with only five complexes selected according to the results of principal component analysis, we were able to predict binding affinities for the other mutants of the particular Ras-effector complex. As the COMBINE analysis method is able to explicitly reveal the amino acid residues that have most influence on binding affinity, it is a valuable aid for protein design.

Ras; RalGDS; Raf; Rap; mutation; binding affinity; binding specificity; Poisson– Boltzmann electrostatics; hot spots; quantitative structure-activity relationship

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