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 γ-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 γ-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 2þ 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.he Ras/Raf/MEK/ERK signaling pathway is the most well studied of five known mitogen activated protein kinase (MAPK) cascades involved in the mediation and timing of signaling events in the cell (1). This pathway is activated by Ras GTPase in response to extracellular signals and is involved in the control of cell proliferation, differentiation, and survival (2). In its resting state Ras is bound to GDP and is in a conformation in which it does not interact with Raf or other effector proteins (3). Guanine nucleotide exchange factors facilitate the release of GDP (4). Once the more abundant GTP binds, the Ras switch I (residues 30-40) and switch II (residues 60-76) regions become poised for interaction with effector proteins, leading to the propagation of signal transduction cascades. Ras has a low intrinsic rate of GTPase activity that is enhanced by 3-5 orders of magnitude in the presence of GTPase activating proteins (GAPs), resulting in depletion of Ras-GTP as the switch is turned off (5).The biochemical properties of Ras and its oncogenic mutants have been well characterized in the absence of Raf or other factors (6, 7), and numerous structures of wild-type and oncogenic Ras mutants have been used to study the possible mechanisms through which Ras becomes defective in its ability to hydrolyze GTP (8-12). The switch regions in these structures, solved from the crystal form with symmetry of space group P3 2 21, are modulated by crystal contacts to resemble the switch I and switch II conformations found in the Ras/RasGAP complex (5, 13). Since the structure of this complex has elucidated the m...

Accessing protein conformational ensembles using room-temperature X-ray crystallography

“…This model makes the prediction that perturbations along the network, such as binding of effectors or mutations along helix 3, could increase conformational coupling to alter the functional properties of H-Ras. Indeed, adventitious Ca 2þ and acetate binding in the allosteric cavity stabilizes the active conformation of the Gln61 network in crystals (34) (Fig. S6C), and a mutation that adds hydrophobic bulk to this cavity (Lys101Leu) switches cellular morphologies associated with Ras activation of the Raf kinase (35).…”

Crystal Structure of the Gtr1pGTP-Gtr2pGDP Protein Complex Reveals Large Structural Rearrangements Triggered by GTP-to-GDP Conversion

“…1D). This tyrosine residue provides a hydrogen bond to the ␥-phosphate group of GTP and is believed to stabilize the transition state complex generated during GTP hydrolysis (17). The corresponding residue in Gtr1p is Leu-38, and it does not interact with GTP (Fig.…”

Crystal structures of the state 1 conformations of the GTP‐bound H‐Ras protein and its oncogenic G12V and Q61L mutants

“…Because association with the various effectors, such as cRaf-1, shifted the equilibrium toward state 2, state 1 and state 2 are regarded as ''inactive'' and ''active'' conformations, respectively [3]. Although crystal structures corresponding to state 2 were solved with wild-type (WT) H-Ras alone or in complex with the effectors [4,5], those corresponding to state 1 have only been solved with its mutants carrying T35S, G60A, and Y32F substitution [6][7][8] [6]. The solution structure of H-RasT35S-GppNHp also revealed polysterism in the switch regions, prominent in switch I [11].…”