Allosteric modulation of Ras positions Q61 for a direct role in catalysis

Abstract

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.

Fig. 1.

The allosteric switch in Ras. Ribbon diagram of Ras-GppNHp showing the shift in helix 3 and loop 7 due to the binding of calcium acetate in the allosteric site. Ras bound to calcium acetate is shown in green with a semitransparent surface. Note the presence of switch II in the model. Ras in the absence of calcium acetate is in yellow (PBD ID code 2RGE). The GppNHp and acetate (with van der Waals dot surface) are shown in stick. (Upper Left) Details of the calcium acetate binding site with a 2Fo-Fc electron density map contoured at the 1σ level. Dashed lines indicate H bonds and calcium ion coordination. All figures were generated in PyMol.

Fig. 2.

H-bonding networks 1 and 2 linking the allosteric site to Q61. The structures in the presence and absence of calcium acetate are shown in green and yellow, respectively. Dashed lines indicate H-bonding interactions involved in the network. (A) Network 1 centered on R97. Shift in helix 3 not visible from this orientation. (B) Network 2 centered on R68, showing the interactions that stabilize switch II. In this orientation the shift in helix 3 is clearly visible. Note the steric overlap between the ordered switch II in the calcium acetate bound structure (green) with helix 3 residues in the structure with an empty allosteric site (yellow). R68 is connected through single water molecules to Y96 (W367) and Q99 (W384) on helix 3, and the carbonyl group of Gly60 (W373) and Q61 (W372) on switch II. All four water molecules are unique to the calcium acetate-bound structure.

Fig. 3.

Allosteric site in structures resulting from soaked crystals. (A) Soak in calcium acetate. (B) Soak in magnesium acetate. (C) Backsoak from magnesium acetate to calcium acetate. The three structures are superimposed on the Ras structure from crystals taken directly from the mother liquor containing calcium acetate, depicted in gray. The 2F_o-F_c electron density maps were contoured at the 1σ level.

Fig. 4.

Proposed mechanism of intrinsic hydrolysis in Ras. (A) Ras-GppNHp active site showing Y32, Q61, the catalytic (W175) and bridging (W189) water molecules near the nucleotide. Electron density (gray) is from a 2Fo-Fc map contoured at the 1σ level. H-bonding interactions involving these residues in the ground state are shown in red dashed lines. The orange dashed line indicates the H bond between W189 and the β-γ oxygen of GTP that we propose helps stabilize the transition state of the reaction. (B) Schematics of the reaction mechanism leading to the transition state. Hydrogen atoms are not shown except those for W175 and W189 and hydrogen atoms that interact directly with them. The flexibility in directionality of H bonds may be important.