Structural basis for GPCR-independent activation of heterotrimeric Gi proteins

Abstract

Heterotrimeric G proteins are key molecular switches that control cell behavior. The canonical activation of G proteins by agonist-occupied G protein-coupled receptors (GPCRs) has recently been elucidated from the structural perspective. In contrast, the structural basis for GPCR-independent G protein activation by a novel family of guanine-nucleotide exchange modulators (GEMs) remains unknown. Here, we present a 2.0-Å crystal structure of Gαi in complex with the GEM motif of GIV/Girdin. Nucleotide exchange assays, molecular dynamics simulations, and hydrogen-deuterium exchange experiments demonstrate that GEM binding to the conformational switch II causes structural changes that allosterically propagate to the hydrophobic core of the Gαi GTPase domain. Rearrangement of the hydrophobic core appears to be a common mechanism by which GPCRs and GEMs activate G proteins, although with different efficiency. Atomic-level insights presented here will aid structure-based efforts to selectively target the noncanonical G protein activation.

Keywords: GIV/Girdin; X-ray crystallography; guanine-nucleotide exchange modulator (GEM); hydrogen–deuterium exchange; molecular dynamics.

Fig. 1.

GIV-GEM binds Sw-II of Gαi. (A) Topology of the Gαi protein with conformational switches and binding sites of key interactors marked. (B) Crystal structure of Gαi with GIV-GEM peptide bound at Switch (Sw)-II. (C) Overlay of Gαi Sw-II from Gβγ-bound, GDI-bound, and GIV-GEM–bound crystal structures. (D) A close-up view of the interaction interface between Gαi and GIV-GEM. (E and F) Close-up views of Gαi Sw-II bound to Gβγ [E, PDB ID code 1GP2 (36)] or GoLoco-motif GDI RGS14 [F, PDB ID code 1KJY (31)]. Key Sw-II residues shared by GIV and at least 1 of Gβγ or RGS14 are shown as spheres (aromatic/aliphatic) or sticks (polar). (G) Bubble plot displaying the strength and the nature of contacts that Gαi Sw-II residues make with GIV-GEM, Gβγ, or RGS14. The size of the dot is proportional to the strength of the contact (57); backbone and side-chains contacts are shown in black and gray, respectively.

Fig. 2.

Structural basis for phosphoregulation of GIV binding and activity toward Gαi. (A) Structure of WT GIV-GEM, highlighting unphosphorylated S1674 and the various contacts of R208^G.H2.4 of Gαi. (B) Model of (pS1674)GIV-GEM highlighting the formation of an additional direct contact with R208^G.H2.4. (C) Structure of WT GIV-GEM, highlighting a polar contact that unphosphorylated S1689 makes with W258^G.H3.17 of Gαi.

Fig. 3.

Homology models of Gαi•GDP bound to the various members of the GEM family suggest a conserved mechanism of binding and action. (A) Sequence alignment of the GEM motifs within human GIV, Daple, and NUCB1 (Calnuc) sequences. (B) Table summarizing previous mutagenesis studies. (C) Crystal structure of GIV-GEM bound to Gαi. (D and E) Homology models of (D) Daple and (E) NUCB1 bound to Gαi created using the GIV-GEM–bound structure as template. Hydrogen bonds explaining the mutagenesis in B are highlighted. (F) Overlay of our GIV-GEM–bound Gαi structure with the EF-hand motif of NUCB1, previously determined by NMR (PDB ID code 1SNL).

Fig. 4.

GIV binding to Sw-II of Gαi disrupts GDP-stabilizing interactions between Sw-II and Sw-I and induces a low-GDP-affinity conformation of Gαi. (A–E) Comparison of Sw-I, Sw-II, and Q204 in various GDP-bound structures of Gαi, arranged from high (Left) to low (Right) GDP-affinity states. In the top part of C, the only two existing structures of GDP-bound monomeric WT Gαi are shown, PDB ID codes 1BOF and 1GDD, both with disordered Sw-II. (F) Overlay of structures shown in A, B, D, and E, highlighting differences in Sw-I and the β2-strand. (G) MANT-GTPγS incorporation into WT and Q204A^G.s3h2.3 Gαi proteins was assessed in the presence of varying concentrations of WT GIV-GEM peptide. Findings are displayed as a line graph showing observed rates (k_obs, s⁻¹) for nucleotide incorporation. Data shown are triplicates from a representative experiment; n = 3. (H) Same data as in G presented as a line graph showing average nucleotide incorporation over time in the presence or absence of 50 μM WT GIV-GEM peptide. Statistical significance between means was calculated using multiple comparisons in one-way nonparametric ANOVA.

Fig. 5.

Bulky hydrophobic residues in Sw-II of Gαi that are engaged by GIV stabilize GDP and influence the dynamics of Sw-I and the β2-strand. (A) Structure showing hydrophobic residues in Sw-II of Gαi that were subjected to mutagenesis. (B and C) MANT-GTPγS incorporation into WT, W211A^G.H2.7, F215A^G.h2s4.1, and V218A^G.h2s4.4 Gαi. Findings are displayed as a dot plot (B) showing the observed nucleotide incorporation rates (k_obs, s⁻¹) and as line graphs (C) showing average nucleotide incorporation over time. Data shown are from three independent experiments; n = 9, 7, 8, and 7 for WT, W211A^G.H2.7, F215A^G.h2s4.1, and V218A^G.h2s4.4, respectively. (D and E) Differences in relative deuterium uptake between V218A^G.h2s4.4 and WT Gαi (D) and between W211A^G.H2.7 and WT Gαi (E) at 5 min, as determined by triplicate HDX-MS assays. Blue and red coloring corresponds to −10% and +10% change, respectively; black indicates regions that were not mapped. Regions exhibiting increased uptake in the W211A^G.H2.7 mutant are highlighted and the corresponding deuterium uptake plots shown (SD error bars are within the symbols). Statistical significance between means was calculated using multiple comparisons in one-way nonparametric ANOVA.

Fig. 6.

Binding of GIV-GEM overcomes the GDP-stabilizing role of Sw-II and releases conformational constraints on Sw-I, β2–β3 strands, and the hydrophobic core of the GTPase domain. (A) Root mean square fluctuations (RMSF, angstroms) of Gαi residues as determined by molecular dynamics simulations under the three specified conditions. (B) Representative histograms showing the distribution, across all simulation frames, of residue rmsd in relation to the mean position of the same residue (F191 in the left and Q204 in the right). (C) Residue RMSF differences between the GIV-GEM–bound Gαi•GDP and Gαi•GDP alone mapped onto the structure of Gαi. (D) Intramolecular distances where the most significant changes between the two simulation conditions (as in C), as determined by PCA, are shown as dotted lines; significant distances beyond the hydrophobic core are colored silver. (E) Representative frames from the MD simulations highlighting the conformational changes allosterically induced by GIV-GEM and perturbation of key interactions in the hydrophobic core of Gαi. (F) Distribution of interresidue distances for the indicated residue pairs throughout the molecular dynamics simulations.

Fig. 7.

GEMs and GPCRs bind at nonoverlapping interfaces on Gαi but both perturb the hydrophobic core of the GTPase domain to stimulate GDP release. (Left) Structure displaying GPCR interface and subsequent Gαi dynamics that ultimately result in GDP release. (Right) Structure displaying GEM interface and subsequent Gαi dynamics that ultimately result in GDP release. Purple color highlights regions of Gαi that move during activation, and yellow arrows describe the direction those regions move. For clarity, only part of the GTPase domain of Gαi is shown.