A P-loop mutation in Gα subunits prevents transition to the active state: implications for G-protein signaling in fungal pathogenesis

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 Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα 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 Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(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 Gα(i1)(G42R) binding to GDP·AlF(4)(-) or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gα(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 Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

Figure 1. A crystal structure of Gα_i1(G42R)·GDP in complex with the phage display peptide KB-752.

(A) The overall structure of Gα_i1 (cyan) with switch regions in dark blue, bound to KB-752 (red) (current study; PDB 3QE0), is overlaid on the wild type Gα_i1·GDP/KB-752 complex (wheat/red transparency) (PDB 1Y3A). GDP is represented by green sticks and magnesium by an orange sphere. (B) The Arg-42 side chain extends from the P-loop, making no polar contacts with other Gα_i1(G42R) residues, but preventing the wild type (transparent) switch conformation. Gα_i1(G42R) residues Arg-178 and Lys-180 are displaced relative to wild type due to steric and electrostatic repulsion by Arg-42. The G42R β2 strand and switch 2 also adopt slightly different conformations. For crystallographic data collection and refinement statistics, see Table S2.

Figure 2. Gα_oA(G42R) is not GTPase deficient, but retains a normal nucleotide cycle and does not interact with RGS domain.

(A) A comparison of radiolabeled GTPγS binding by wild type Gα_oA (k_on = 0.087±0.020 min⁻¹ (s.e.m.)) and Gα_oA(G42R) (k_on = 0.062±0.010 min⁻¹ (s.e.m.)) identified no significant difference in the rate of GDP release and subsequent GTP analog binding. (B) Gα_oA(G42R) retained the ability to hydrolyze GTP (k_cat = 0.18±0.05 min⁻¹ (s.e.m.)) at a rate indistinguishable from wild type Gα_oA (k_cat = 0.19±0.02 min⁻¹ (s.e.m.)), as determined by single turnover hydrolysis assays. (C) Surface plasmon resonance (SPR) experiments demonstrated selective binding of the transition state-mimetic, GDP·AlF₄ ⁻-bound form of Gα_oA to the RGS domain of RGS12. Gα_oA(G42R) did not interact with the RGS12 RGS domain in any nucleotide state at concentrations up to 25 µM (D). An equilibrium binding isotherm allowed quantification of wild type Gα_oA affinity for RGS12 (K_D = 1.27±0.06 µM (s.e.m.)).

Figure 3. Gα_i1(G42R) engages inactive conformation-selective binding partners in two nucleotide states.

(A) Wild type Gα_i1 binds Gβ₁γ₁ only in the GDP-bound state, as determined by SPR, while Gα_i1(G42R) displayed no nucleotide state-selectivity of Gβ₁γ₁ binding when liganded with either GDP or GDP·AlF₄ ⁻. (B) Similarly, fluorescence polarization experiments showed highly nucleotide state-selective binding of the RGS14 GoLoco motif to wild-type Gα_i1·GDP (K_D = 9.0±1.1 nM (s.e.m.)) compared to the AlF₄ ⁻-bound form (K_D = 8.7±1.0 µM (s.e.m.)), but both nucleotide states of Gα_i1(G42R) interacted with the GoLoco motif peptide, with affinity constants of 45±7 nM (s.e.m.) and 168±27 nM (s.e.m.) for GDP and AlF₄ ⁻, respectively. (C) The activated state-selective peptide KB-1753 preferentially bound the AlF₄ ⁻-bound form of wild-type Gα_i1 (K_D = 470±40 nM (s.e.m.)) compared to the GDP-bound form (K_D = 6.7±0.4 µM (s.e.m.)), but had low affinity for Gα_i1(G42R) in both nucleotide states.

Figure 4. The G42R point mutation prevents Gα_i1 from assuming the activated conformation.

Upon binding GDP·AlF₄ ⁻, the switch regions of Gα_i1 undergo a conformational change, burying the switch 2 Trp-211 in a hydrophobic cleft . As a result, the intrinsic tryptophan fluorescence of Gα_i1 increases, and the activated switch conformation is protected from trypsin proteolysis, relative to the GDP-bound state. (A) The intrinsic tryptophan fluorescence of wild type Gα_i1 increased upon injection of AlF₄ ⁻, while the response of Gα_i1(G42R) was blunted. (B) Gα_i1 was relatively resistant to trypsin proteolysis upon loading with either GDP·AlF₄ ⁻ or GTPγS. In contrast, Gα_i1(G42R) was efficiently proteolyzed in any nucleotide state. (C) The Gα_i1(G42R)·GDP/RGS14 GoLoco crystal structure model of this study (PDB 3QI2) is shown in cyan with the Arg-42 side chain in magenta sticks. GDP and magnesium are represented as green sticks and an orange sphere, respectively. The GoLoco motif peptide is excluded for clarity. For a complete model, see Figure S2. (D) The activated, GTPγS-bound form of wild type Gα_i1 (PDB 1GIA) is shown in gray. Upon binding to the GTP analog, the switch regions (SI-III) of wild type Gα_i1 converge on the phosphoryl groups of the nucleotide, resulting in a conformation recognized by effector molecules. However, the mutant Arg-42 side chain extending from the P-loop (superposed in magenta) is not sterically accommodated in a wild type-like activation state; switch 3 residues Leu-234 and Glu-236 would clash with the mutant residue. Thus, Arg-42 does not allow Gα_i1(G42R) to assume a typical active conformation, although the critical residues Glu-204 and Arg-178 apparently can be positioned for efficient GTP hydrolysis (see Fig. 2).

Figure 5. Gα_q G48R is not constitutively active in a cellular context.

The analogous P-loop mutation in human Gα_q, G48R, did not yield constitutive activity in contrast to the GTPase-deficient Gα_q(Q209L) (A,B). Transfection of increasing amounts of Gα_q(Q209L) markedly stimulated phospholipase C (PLC) activity in COS-7 cells, indicated by increased inositol phosphates (IP_x) accumulation. Like wild type Gα_q, G48R overexpression did not alter PLC activity. (C,D) Endogenous and overexpressed KT3 epitope-tagged wild type Gα_q stimulated PLC activity upon treatment with AlF₄ ⁻. The response of cells expressing Gα_q(G42R) was blunted relative to wild type Gα_q.

Figure 6. M. oryzae strains expressing G42R or GTPase-deficient Q204L mutant Gα subunits show disparity in appressoria formation.

(A) Conidia harvested from the magA^G45R, magA^Q208L and WT strains were inoculated on inductive (plastic cover slips) or non-inductive surfaces (GelBond membrane) and assessed for the ability to form appressoria after 16 hpi (hours post inoculation). The 2-celled conidia (white arrow) of the magA^Q208L produced aberrant appressorium (white asterisk) on both inductive and non-inductive surfaces. Insets represent the highly pigmented structures (black arrowhead) made by the magA^Q208L strain. Scale bars = 10 µm. (B) Bar graph illustrating the efficiency of appressorium formation in the magA^G45R, magA^Q208L and wild type strains on inductive (black bar) or non-inductive surfaces (gray bar) respectively. Values represent mean ± S.E from three independent replicates involving 300 conidia per sample. (C) Identical experiments were conducted on the corresponding magB wild type and mutant strains. Unlike the wild type, the majority of conidia from the magB^G42R strain failed to produce melanized appressoria efficiently on inductive surfaces. A small proportion of the magB^G42R conidia produced mature appressoria on the non-inductive surface (indicated by the white arrow). Conidia from the magB^Q204L failed to produce appressoria on both inductive and non-inductive surfaces. (D) Bar graph showing quantification of appressorium formation, as in (B).

Figure 7. Expression of non-activatable (G42R) or GTPase-deficient (Q204L) Gα subunits differentially affects M. oryzae pathogenicity.

Barley leaf explants were spot inoculated in triplicate with the specified number of conidia (500, 100 and 2000 per inoculation site) from the magA^G45R, magA^Q208L, magB^G42R, magB^Q208L and wild type strains and the disease symptoms scored 7d post inoculation. The magA^G45R caused typical disease lesions comparable to the wild type. The magA^Q208L failed to cause typical blast lesions even at high spore counts. The magB^G42R caused mild disease lesions on barley leaf explants inoculated with higher concentration of spores. Under comparable conditions, conidia from the magB^Q208L were incapable of causing disease.