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. 2019 May 16;177(5):1232-1242.e11.
doi: 10.1016/j.cell.2019.04.022. Epub 2019 May 9.

Assembly of a GPCR-G Protein Complex

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Free PMC article

Assembly of a GPCR-G Protein Complex

Yang Du et al. Cell. .
Free PMC article

Abstract

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Keywords: G protein; G protein-coupled receptor; conformation; dynamics; hydrogen/deuterium exchange mass spectrometry; hydroxyl radical footprinting mass spectrometry.

Conflict of interest statement

DECLARATION OF INTERESTS

Brian Kobilka is a co-founder of and a consultant for ConformeRx, Inc.

Figures

Figure 1.
Figure 1.. Structures representing different stages in the G protein cycle
(A) GPCR-mediated G protein activation illustrated with representative crystal structures. Left: The GDP-bound heterotrimeric G protein (Liu et al., companion manuscript) shows the position of nucleotide-binding pocket. GDP is shown as a stick model with carbons colored green. The Ras-like domain of Gα is colored as light orange, the α-helical domain (AHD) of Gα as light yellow, Gβ as light blue, and Gγ as violet. Middle: The X-ray crystal structure of the β2AR-Gs complex (PDB: 3SN6) shows a large movement of AHD and opening of the nucleotide-binding pocket. The β2AR is shown in grey. Nanobody Nb35 and the T4 lysozyme insertion that were essential for crystallization have been omitted for clarity. Right: The GTP-bound Gα subunit structure (PDB: 1AZT) shows the position of GTP (stick model) and closing of the nucleotide binding pocket. (B) Comparison of the high-resolution X-ray crystal structures of the β2AR in an agonist-bound form (PDB: 3PDS, cyan) and an agonist-bound Gs-coupled form (PDB: 3SN6, grey). The nucleotide-free state of Gα is shown in light orange. (C) Comparison of the X-ray crystal structures of the Ras-like domain of Gαs in a GDP-bound form (Liu et al., companion manuscript, light blue) and a β2AR-bound nucleotide-free form (PDB: 3SN6, light orange). The active state of the β2AR (PDB: 3SN6) is shown in grey. (D) Description of the ICL2 and the cytosolic-core links in the nucleotide-free GPCR-G protein complex. Receptor binding signals through the Ras-like domain of Gα to the nucleotide-binding pocket (blue) via the ICL2 (green) and the cytosolic-core (red) links. GDP (stick model) is positioned in the nucleotide-free structure of the β2AR-Gs complex based on structural alignment with the GDP-bound heterotrimeric Gs protein (Liu et al., companion manuscript). The rest of the Ras-like domain of Gα subunit is shown in light orange and the β2AR in grey.
Figure 2.
Figure 2.. Time-resolved analysis of GPCR-Gs complex formation by HDX-MS
(A) GDP release was complete within 10 sec of incubation with BI-167107-bound β2AR. (B–D) HDX profiles of selected peptides from the β2AR (B) and Gαs (C and D). The analyzed peptides are indicated as colored regions on the X-ray crystal structure of the β2AR-Gs complex (PDB: 3SN6). The HDX profile changes of the β2AR upon incubation with Gs at the room temperature are analyzed by a 10 sec D2O pulse (B). HDX profile changes of Gαs upon co-incubation with the β2AR or A2A at the room temperature (C) or on ice (D) were analyzed with 10 sec or 100 sec D2O pulses. Statistical significance of the incubation time dependent HDX changes were analyzed by repeated measures ANOVA (rANOVA), and the results from rANOVA analysis are presented in Table S1. To compare time points, a paired t-test was used and p<0.05 was considered to be statistically significant. *, the first incubation time point that shows statistical difference from the β2AR alone or the GDP-bound state of Gs. #, the first incubation time point that shows statistical difference from the * time point. +, the first incubation time point that shows statistical difference from the # or previous + time point. Error bars represent the s.e.m. Please note that the data is plotted using a non-linear/non-logarithmic scale. See also Figure S1–4 and Table S1.
Figure 3.
Figure 3.. Time-resolved analysis of GPCR-Gs complex formation by HRF-MS
(A) X-ray generated radiolytic oxidative modification profiles of selected peptides or residues from the β2AR or Gαs. Oxidative modification changes of Gαs upon incubation with the β2AR were analyzed. The modified peptides or residues are indicated as colored regions or sticks on the X-ray crystal structure of the β2AR-Gs complex (PDB: 3SN6). Statistical significance of the incubation time dependent changes were analyzed by rANOVA, and the results from rANOVA analysis are presented in Table S1. To compare time points, a t-test was used and p<0.05 was considered to be statistically significant. *, the first incubation time point that shows statistical difference from the β2AR alone or the GDP-bound state. #, the first incubation time point that shows statistical difference from the * time point. Error bars represent the s.e.m. The time series of M386 (381–394) is not significant by rANOVA (p=0.110), but t-test showed that 800 ms is significantly different from initial 20 ms (p=0.03). This is not to say that the t-test is incorrect, only that the entire series is too variant in spread at each time point to support a statement that the entire series is significant to a reasonable α value. (B) The surrounding environment of M386. In the GDP-bound Gs structure, M386 is located within a pocket formed by four amino acids (green spheres) with limited solvent exposure (C) Rearrangement of interactions with M221 and F376 of Gas following formation of the nucleotide-free β2AR-Gs complex. In the GDP-bound Gs structure, M221 and F376 form interactions with residues within β2-β3 strands and α1 helix (left), which are lost in the β2AR-bound nucleotide-free structure (PDB: 3SN6) (right). In the β2AR-bound nucleotide-free structure (PDB: 3SN6), F376 forms new interactions with F139 of the β2AR and amino acids in the αN/β1 hinge and β2/β3 loop (right). See also Figure S1 and Table S1.
Figure 4.
Figure 4.. Functional analysis of the roles of Gαs C-terminus and β2AR ICL2
(A–C) Effect of Gαs C-terminal five-residue truncation (Gs_∆5) on β2AR-induced GDP release activity (A), Gs-stabilized movement of TM6 of the β2AR (B), and agonist affinity change (C). The C-terminal five-residue truncation mutant failed to release GDP upon co-incubation with the β2AR (A), failed to induce a bimane fluorescence change (B), and failed to stabilize the high-affinity agonist-binding state in the β2AR (C). (D–F) Effect of β2AR F139A mutation on β2AR-induced GDP release from Gs (D), Gs-induced movement of TM6 of the β2AR (E), and agonist affinity change (F). β2AR F139A cannot catalyze the release of GDP from Gs (D). Changes in bimane fluorescence (B) and agonist affinity (E) suggest that β2AR F139A forms a ternary complex with Gs, but the conformation is different from that observed with the wild-type β2AR. The data represent the mean ± SD of three independent measurements. Statistical significance of data in Figures 4A, 4B, 4C, and 4E were analyzed by one-way ANOVA followed by Tukey’s post-test, Figure 4D by rANOVA followed by Tukey’s post-test, and Figure 4F by unpaired t-test. *p value of <0.01. Figures 4B and 4E are representative traces from three independent experiments.
Figure 5.
Figure 5.. Proposed model for Gs activation by the β2AR.
(A) Summary cartoon illustrating proposed sequence of events during GPCR-Gs complex formation and GDP release from Gα subunit. Ras indicates Ras-like domain and AHD indicates α-helical domain. (B) Intra molecular interactions between αN/β1 hinge and α5 in the GDP-bound Gs. R389 in and E392 are solvent exposed and available for interactions with agonist-bound β2AR. (C) Effect of Nb37 on the kinetics of GTPγS-induced complex dissociation assayed by bimane fluorescence. The data shows a representative of three independent experiments. (D) Effect of Nb37 on the kinetics of BODIPY-FL GTPγS binding of the β2AR-Gs complex. The data shows a representative of three independent experiments.

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