Transition State Mimic of Intrinsic Hydrolysis in Ras GTPase

Abstract

Ras GTPases are involved in signal transduction pathways that control fundamental cellular processes such as proliferation, cycling, survival, and apoptosis. Ras functions via an on/off switch mechanism, where Ras is active in downstream signaling when bound to GTP and inactive upon GTP hydrolysis to GDP. Our lab discovered an allosteric site that is proposed to interact with the membrane, where binding in this allosteric site promotes ordering of the switch II region, favoring a conformation conducive to hydrolysis of GTP to GDP. When Ca2+ and acetate bind at this site in Ras crystals there is a disorder to order transition in switch II, positioning catalytic residues in place for hydrolysis. Interestingly, mutations of R97 to bulky aliphatic residues, such as L and M, mimic this effect, increasing hydrolysis rate constants relative to wild type. We had previously shown that in the presence of AlF3 RasR97L‐GDP binds to the Ras binding domain of Raf (Raf‐RBD), mimicking the γ‐phosphate of GTP. Here we present gel filtration studies of the R97M‐GDP mutant in the presence of Raf and AlF3 showing complex formation. The fact that Raf‐RBD binds to the R97L and R97M mutants, but not to wild type Ras, suggests that in these mutants the conformation associated with hydrolysis is more populated than in the wild type. AlFx has been used extensively to visualize the transition state of the GAP‐catalyzed GTP hydrolysis on Ras and its homologues. We are currently working on obtaining crystals of the H‐RasR97M‐Raf‐AlFx complex as a transition state model to elucidate the mechanism of intrinsic hydrolysis in Ras.Support or Funding InformationNSF MCB‐1517295