An allosteric site mutant of Ras stabilizes a catalytically active conformation of switch II

Ras is a small GTPase which propagates signaling along the Ras/Raf/MEK/ERK pathway when bound to GTP. This pathway is up‐regulated in many human cancers. Recently, our lab solved the structure of Ras bound to GppNHp with an acetate ion interacting with Helix 3 residue R97 at an allosteric site. The presence of calcium acetate at this site results in a shift in Helix 3/loop 7 and a highly ordered switch II. Thus, the allosteric site modulates Q61, a residue important for hydrolysis of GTP to GDP in Ras. With the intent of disrupting the allosteric modulation of switch II we generated the R97L mutant, mimicking the approximate size and aliphatic portion of the wild type without the positive charge essential for interaction with the acetate. The crystal structure of the RasR97L‐GppNHp shows an “empty” allosteric site, but the switch II is ordered as in the activated form of the wild type. The Helix 3/loop 7 shift is stabilized by hydrophobic interactions between L97 and Y137. Thus, the R97L mutation shifts the conformational equilibrium toward the “on” state of the allosteric switch, with an ordered switch II poised for catalysis. This is supported by a two‐fold increase in hydrolysis rate of the mutant relative to wild type and the fact that RasR97L‐GDP binds Raf RBD in the presence of the transition state mimic AlF 3 . This is not the case for wild type Ras‐GDP, which primarily samples the “off” state with a disordered active site that cannot bind AlF 3 and thus remains in the GDP‐bound state that does not bind Raf. Our analysis of RasR97L supports our previously proposed GAP‐independent mechanism of intrinsic hydrolysis in Ras, modulated by the allosteric switch in the Ras/Raf complex.