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Originally published In Press as doi:10.1074/jbc.M101727200 on May 1, 2001

J. Biol. Chem., Vol. 276, Issue 29, 27629-27637, July 20, 2001
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Structure-based Mutagenesis Reveals Distinct Functions for Ras Switch 1 and Switch 2 in Sos-catalyzed Guanine Nucleotide Exchange*

Brian E. HallDagger , Shao Song Yang§, P. Ann Boriack-Sjodin||, John Kuriyan, and Dafna Bar-Sagi**Dagger Dagger

From the ** Department of Molecular Genetics and Microbiology and the Graduate Programs in Dagger  Molecular Pharmacology and § Molecular and Cellular Biology, State University of New York at Stony Brook, New York 11794-5222 and the  Laboratories of Molecular Biophysics and the Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021

Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates 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-directed mutagenesis to investigate the functional significance of the conformational 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 coordinated 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 important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras·Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg2+- and nucleotide phosphate-binding site of switch 2 (Ras Ala59 and Sos Leu938 and Glu942) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr32 and Tyr40) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.

* This work was supported by National Institutes of Health Grant CA28146 and by the Carol Baldwin Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Present address: Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142.

Dagger Dagger To whom correspondence should be addressed: Dept. of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Life Science Bldg., Stony Brook, NY 11794-5222. Tel.: 631-632-9737; Fax: 631-632-8891; E-mail: barsagi@pharm.sunysb.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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