Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal
transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras
and are found prominently in human cancers. Yet the mechanism through which Q61
contributes to catalysis has been elusive. It is thought to position the
catalytic water molecule for nucleophilic attack on the gamma-phosphate of GTP.
However, we previously solved the structure of Ras from crystals with symmetry
of the space group R32 in which switch II is disordered and found that the
catalytic water molecule is present. Here we present a structure of wild-type
Ras with calcium acetate from the crystallization mother liquor bound at a site
remote from the active site and likely near the membrane. This results in a
shift in helix 3/loop 7 and a network of H-bonding interactions that propagates
across the molecule, culminating in the ordering of switch II and placement of
Q61 in the active site in a previously unobserved conformation. This structure
suggests a direct catalytic role for Q61 where it interacts with a water
molecule that bridges one of the gamma-phosphate oxygen atoms to the hydroxyl
group of Y32 to stabilize the transition state of the hydrolysis reaction. We
propose that Raf together with the binding of Ca(2+) and a negatively charged
group mimicked in our structure by the acetate molecule induces the ordering of
switch I and switch II to complete the active site of Ras.
