A P-loop mutation in G? subunits prevents transition to the active state: Implications for G-protein signaling in fungal pathogenesis
Bosch D.E. Willard F.S. Ramanujam R. Kimple A.J. Willard M.D. Naqvi N.I. Siderovski D.P.
Bosch D.E.
Willard F.S.
Ramanujam R.
Kimple A.J.
Willard M.D.
Siderovski D.P.
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Alternative Title
Abstract
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??? heterotrimer relies on nucleotide cycling by the G? subunit: exchange of GTP for GDP activates G?, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting G? 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? subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that G?(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?i1(G42R) binding to GDP·AlF4- or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. G?(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with G?? and GoLoco motifs in any nucleotide state. The corresponding G?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? subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two G? mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants. © 2012 Bosch et al.
Keywords
aluminum derivative, aluminum tetrafluoride, arginine, G protein coupled receptor, guanine nucleotide binding protein, guanosine triphosphatase, kb 1753, unclassified drug, guanine nucleotide binding protein alpha subunit, mutant protein, article, binding affinity, binding site, controlled study, crystal structure, DNA binding motif, fungal strain, fungus, gene locus, gene mutation, hydrolysis, intracellular signaling, nonhuman, pathogenesis, protein conformation, protein protein interaction, signal transduction, amino acid substitution, chemical structure, chemistry, enzyme active site, genetics, Hordeum, Magnaporthe, metabolism, microbiology, mycosis, pathogenicity, physiology, plant disease, plant leaf, point mutation, protein folding, protein tertiary structure, signal transduction, X ray crystallography, Fungi, Magnaporthe grisea, Magnaporthe oryzae, Thespesia grandiflora, Amino Acid Substitution, Catalytic Domain, Crystallography, X-Ray, GTP-Binding Protein alpha Subunits, Hordeum, Magnaporthe, Models, Molecular, Mutant Proteins, Mycoses, Plant Diseases, Plant Leaves, Point Mutation, Protein Folding, Protein Structure, Tertiary, Signal Transduction
Source Title
PLoS Pathogens
Publisher
Series/Report No.
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Rights
Attribution 4.0 International
Date
2012
DOI
10.1371/journal.ppat.1002553
Type
Article