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...
The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
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