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...
Summary Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.
Background:The GTP nucleotide is thought to be fully deprotonated when bound to RAS. Results: The neutron crystal structure of RAS bound to the GTP analogue GppNHp shows a protonated ␥-phosphate. Conclusion: The active site of RAS significantly increases the pK a of the nucleotide. Significance: This work provides insight to the GTP hydrolysis mechanism, with implications to the superfamily of small GTPases.
Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.
CD4 and chemokine receptors mediate HIV-1 attachment and entry. They are, however, insufficient to explain the preferential viral infection of central memory T cells. Here, we identify L-selectin (CD62L) as a viral adhesion receptor on CD4+ T cells. The binding of viral envelope glycans to L-selectin facilitates HIV entry and infection, and L-selectin expression on central memory CD4+ T cells supports their preferential infection by HIV. Upon infection, the virus downregulates L-selectin expression through shedding, resulting in an apparent loss of central memory CD4+ T cells. Infected effector memory CD4+ T cells, however, remain competent in cytokine production. Surprisingly, inhibition of L-selectin shedding markedly reduces HIV-1 infection and suppresses viral release, suggesting that L-selectin shedding is required for HIV-1 release. These findings highlight a critical role for cell surface sheddase in HIV-1 pathogenesis and reveal new antiretroviral strategies based on small molecular inhibitors targeted at metalloproteinases for viral release.
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