Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Pas and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in het erotrimeric G proteins. Aluminum. fluoride activates the GDP form of hetero trimeric G proteins, and enhances binding of the GDP form of small G protei ns to their GAPs. The resultant complexes have been interpreted as analogue s of the transition state of the hydrolytic reaction. Here, equilibrium bin ding has been measured using scintillation proximity assays to provide quan titative information on the fluoride-mediated interaction of Ras and Rile p roteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-af finity fluoride-mediated complex formation between Rho . GDP and RhoGAP occ urred in the absence of aluminum; however, under these conditions, magnesiu m was required. Additionally, the novel observation was made of magnesium-d ependent, fluoride-mediated binding of Ras . GDP to NF1 in the absence of a luminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum:fluoride or magnesium fluoride can mediate the high-affinity binding of Rho . GDP or Ras . GDP to GAPs. I t has been reported that magnesium fluoride can activate heterotrimeric G p roteins. Thus, magnesium-dependent fluoride effects might be a general phen omenon with G proteins. Moreover, these data-suggest that some protein nucl eotide complexes previously reported to contain aluminum fluoride may in fa ct contain magnesium fluoride.