Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange

Ras GTPases function as binary switches in signaling pathways controlling c ell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We hav e previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P.A., Margarit, S.M., Ba r-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343), The structure demo nstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2, In this study, we have employed site-direc ted mutagenesis to investigate the functional significance of the conformat ional changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordin ated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most im portant contact being mediated by tyrosine 64, which is buried in a hydroph obic pocket of Sos in the Ras Sos complex. Substitutions of Ras and Sos sid e chains that are inserted into the Mg2+- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interact ion of Sos with switch 2 is necessary for tight binding, but is not the cri tical driving force for GDP displacement. The structural distortion of swit ch 1 induced by Sos is mediated by a small number of specific contacts betw een highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intr insic rate of nucleotide dissociation from Ras and impair the binding of Ra s to Sos. Based on this analysis, me propose that the interactions of Sos w ith the snitch 1 and switch 2 regions of Ras have distinct functional conse quences: the interaction with switch 2 mediates the anchoring of Ras to Sos , whereas the interaction with switch 1 leads to disruption of the nucleoti de-binding site and GDP dissociation.