Structural and functional analysis of Ras cycling and inhibition

Ras is a small guanine nucleotide binding protein ("G protein") that serves as a molecular switch by cycling between GDP- and GTP-bound states in response to stimulation of receptor tyrosine kinases. The 20kDa Ras core is the prototype for all other monomeric ("small") G proteins as well as the alpha subunit of heterotrimeric G proteins and others. The work in this dissertation investigates the role of conserved structural motifs in producing physiological and pathological Ras signaling and assesses ways that oncogenic Ras may be inhibited.; "Dominant negative" Ras mutants such as RasS17N, G60A and others share a common ability to block signaling through normal or pathologically active Ras proteins in vivo. We show that mutation of Ras glycine 60 to alanine produces a form of Ras that, when bound to GTP, adopts an "open" conformation reminiscent of that of nucleotide-free Ras in complex with the nucleotide exchange factor SOS. We further show that RasG60A-GTP forms a stable complex with SOS and propose that RasG60A behaves as a dominant negative through formation of a stable and unproductive Ras/nucleotide/SOS complex and by loss of binding to Raf kinase. We further demonstrate that mutation of Ras glutamine 61 to glycine produces an entirely different biochemical and structural outcome.; Additionally, we present the structure of the G60A/K147A double mutant of Ras, which, along with the GTP-bound form of RasG60A, shows characteristics consistent with those of a structural intermediate for nucleotide exchange. Finally, we use the mutants above, the structure of a RasG60A-GppNp/RafRBD complex and the RasE37A mutant to demonstrate that, in solution, wild-type Ras interconverts between "GTP", "GDP" and "open" conformations.; Overall, these studies illustrate that Ras depends on dynamic fluctuations in its "switch" regions in order to achieve nucleotide hydrolysis, nucleotide exchange, and effector binding. These fluctuations depend on a finely-balanced interplay between structural elements and flexibility in Ras' 57DxxG 60 motif in switch II, which coordinates conformational states with switch I via a glutamic acid residue at position 37.