A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active G alpha beta gamma heterotrimer relies on nucleotide cycling by the G alpha subunit: exchange of GTP for GDP activates G alpha, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G alpha to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of G alpha subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G alpha(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon G alpha(i1)(G42R) binding to GDP center dot AlF4(-) or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G alpha(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G beta gamma and GoLoco motifs in any nucleotide state. The corresponding G alpha(q)(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the G alpha subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G alpha mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.