Structural and functional analysis of the Ras molecular switch

Ras GTPases function as essential molecular switches in signaling pathways controlling cell proliferation and differentiation by cycling between an active GTP-bound conformation and an inactive GDP-bound conformation. The crystal structures of Ras-GDP and Ras-GTP indicate that the major conformational changes occur in two loop regions termed switch 1 and switch 2.; Ras proteins are activated by the guanine nucleotide exchange of GTP for GDP. This reaction proceeds through a nucleotide-free intermediate that can be stabilized by guanine nucleotide exchange factors (GEFs). We show that nucleotide exchange of Ras, both intrinsic and catalyzed by the Sos GEF, is driven by the displacement of critical switch 1 region residues from the nucleotide binding site. Additionally, we demonstrate that formation of a stable Ras-Sos complex is dependent on hydrophobic interactions between Sos and the switch 2 region of Ras. The nature of this interaction has allowed us to begin developing a system for obtaining mutants of Ras with increased affinity for Sos by monovalent phage display of Ras. Ultimately, these mutants will be used to understand structural determinants for high affinity binding to Sos to provide a structural basis for blocking its activation of Ras.; Ras proteins are inactivated by GTP hydrolysis and the resulting conformational changes. In contrast to the activating GDP to GTP switch which requires nucleotide destabilization and dissociation, the inactivating GTP to GDP switch necessitates the release of only the hydrolyzed inorganic phosphate. We present the structure of a Ras mutant in a conformation strikingly similar to intermediates in the GTP to GDP conformational change predicted by molecular dynamics simulations. The most remarkable feature of the intermediate structure is direct hydrogen bonding between the switch 1 and switch 2 regions. These interactions function to prevent switch 1 displacement and ultimately stabilize the bound nucleotide. Nucleotide stabilization prevents guanine nucleotide exchange, both intrinsic and Sos-catalyzed, during inactivation.; Overall, the studies presented here demonstrate that although Ras guanine nucleotide exchange and GTP hydrolysis are opposite in nature, both processes function by altering nucleotide stability through divergent intermediate structures that use identical regions and even identical amino acids to proceed to completion.