Conformational Exchange and Ligand Binding in Ras

Small GTP hydrolases (GTPases) provide essential signaling capabilities for the cell and are homologous to the alpha subunit of heterotrimeric G proteins. GTPases are considered protein molecular switches that respond to the needs of the cell by alternating between GTP- and GDP- bound states. The G-domain of GTPases has the enzymatic function of hydrolyzing GTP. As part of cell-signaling networks, the &;The Ras GTPase-dependent signaling pathways are notable for their impact on cells and organisms when they are permanently activated. Mutations that prevent Ras•GTP turnover leave the protein effector-competent and unable to terminate signaling. As a potent signal transducer for cell proliferation, Ras has been implicated in human disease, most notably in cancer, where Ras mutations are common.;The &;An oncogenic mutation was introduced to determine the extent of conformational exchange in H-Ras(G12D)•GTP. Our results confirmed that Ras•GTP undergoes a similar global conformational exchange process in comparison with Ras•GppNHp. The addition of GTP and magnesium increased detection of conformational exchange, consistent with a ligand-induced population shift. The identity of the minor state was discussed for a structure of Ras resembling a regulatory protein-bound form.;The molecular switch model for Ras based on the GTPase cycle was reconsidered in light of new insights into Ras activation. A hypothetical reaction coordinate is offered that incorporates the effect of magnesium and GTP on Ras protein dynamics.;We also characterized a non-canonical ligand-binding site of unknown function on the surface of Ras. This remote site, originally discovered by X-ray crystallography in H-Ras•GppNHp, accommodates positively charged ligands. The negatively-charged pocket bound divalent cations magnesium and calcium with millimolar dissociation constants. Unexpectedly, Ras•PGDP displayed higher affinity for ions compared to Ras•PGppNHp, although the weak interaction suggests calcium is not likely a biologically relevant ligand. Magnesium may be a ligand in the cell, although structural comparisons with Ras bound to effectors and regulatory enzymes suggest a larger ligand, perhaps associated with the plasma membrane.;Analysis of binding indicated that the pocket carried an overall charge of -2 in the unliganded form, and -1 when bound to a calcium acetate ion-pair. The remote site is allosterically coupled to the active site, located more than 20 Å away from bound nucleotide. We conclude this site is a general feature on the surface of Ras, present in Ras•GDP and Ras•GppNHp, and that it may be found in Ras bound to effectors, exchange factors, and activating proteins.;It is our current view that the G domain is allosteric, and that membrane interactions may be sensed at the active site, producing changes in protein function. Our studies of conformational exchange demonstrate one possible mechanism for mediating an allosteric change, while our study of ligand binding clearly reveal that an allosteric linkage does exist between the active site and membrane-facing remote site.