Allosteric modulation of Ras: a novel role for Glutamine 61 in catalysis

Abstract

The Ras GTPase and its effector Raf are key mediators of the Ras/Raf/MEK/Erk signal transduction pathway, which plays a central role in cell growth and malignant transformation. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. We have found that Ras‐GppNHp in the crystal binds calcium acetate from the crystallization mother liquor at a site remote from the active site and most likely near the membrane, resulting in a shift in helix 3/loop 7 and a network of H‐bonding interactions that propagates across the molecule, culminating in complete ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation for catalysis. In this structure Q61 interacts with a water molecule that bridges one of the γ‐phosphate oxygen atoms to the hydroxyl group of Y32. This active site arrangement suggests a novel role for Q61 where it interacts with the bridging water molecule to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca2+ and negatively charged group in the membrane at the allosteric site (mimicked in our structure by the acetate molecule), induce ordering of the switch I and switch II to complete the active site and bring intrinsic hydrolysis rates in Ras to biologically relevant levels. Funded by NIH grant CA096867.