ELECTROSTATIC CONTROL OF GTP AND GDP BINDING IN THE ONCOPROTEIN P21(RAS)

Background: p21(ras) is one of the GTP-binding proteins that act as in tercellular molecular switches, The GTP-bound form of p21(ras) sends a growth-promoting signal that is terminated once the protein is cycled back into its GDP-bound form. The interaction of guanine-nucleotide-e xchange factors (GEFs) with p21(ras) leads to activation of the protei n by promoting GDP-->GTP exchange, Oncogenic mutations of p21(ras) tra p the protein in its biological active GTP-bound form, Other mutations interfere with the activity of GEF. Thus, it is important to explore the structural basis for the action of different mutations. Results: T he crystal structures of p21(ras) are correlated with the binding affi nities of GTP and GDP by calculating the relevant electrostatic energi es, It is demonstrated that such calculations can provide a road map t o the location of 'hot' residues whose mutations are likely to change functional properties of the protein. Furthermore, calculations of the effect of specific mutations on GTP and GDP binding are consistent wi th those observed, This helps to analyze and locate functionally impor tant parts of the protein. Conclusions: Our calculations indicate that the protein main chain provides a major contribution to the binding e nergies of nucleotides and probably plays a key role in relaying the e ffect of GEF action. Analysis of p21(ras) mutations in residues that a re important for the proper function of GEFs suggests that the region comprising residues 62-67 in p21(ras) is the major OFF-binding site, T his analysis and our computer simulations indicate that the effect of GEF is probably propagated to the P-loop (residues 10-17) through inte raction between Gly60 and Gly12, This then reduces the interaction bet ween the main-chain dipoles of the P-loop and the nucleotide. Finally, the results also suggest a possible relationship between the GTP-->GD P structural transition and the catalytic effect of the GTPase-activat ing protein.