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COMBINE Analysis of Interactions Between Ras Proteins and 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 interaction in which these proteins are involved is of importance for studying signalling mechanisms and because of their medical relevance as targets for anticancer therapy. We investigated interaction between the Ras proteins (Ras and Rap) and their effectors (Raf and RalGDS) using the COMparative BINding Energy (COMBINE) analysis1. The available crsytal structures of Ras protein with E31K mutant of Ral and Rap protein with Raf protein were used as the starting structures for in silico prepariation of the 122 different wild-type and mutant complexes. COMBINE models were built using chemometrical analysis by which eletrostatic and Van der Waals contributions to the interaction energy and electrostatic binding free energy were correlated to the experimentaly obtained binding free energies2. The COMBINE models highlighted amino acid residues responsible for differences in binding of the same effector to the two different Ras proteins, as well as differences in the binding of the two different effectors (RalGDS and Raf) to Ras. 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. 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. The method used in this study is valuable aid for protein design. 1 S. Tomić, B. Bertoša, T. Wang, R. C. Wade, PROTEINS: Structure, Function, and Bioinformatics, 67 (2) (2007) 435-447. 2 Kiel C, Serrano L, Herrmann C. A., J. Mol. Biol. 2004 ; 340:1039-1058.

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

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