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Universal allosteric mechanism for Gα activation by GPCRs

Nature volume 524, pages 173–179 (2015)Cite this article

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Abstract

G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR–Gα system diversified rapidly, while conserving the allosteric activation mechanism.

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Figure 1: Gα signalling states and activation.
Figure 2: The common Gα numbering (CGN) system and Gα conserved contact networks.
Figure 3: Helix 5 contains the conserved interface region and is comprised of two modules.
Figure 4: Helix H1 is the key SSE that contacts H5, GDP and the H-domain.
Figure 5: Mutational studies support the universal Gα activation mechanism.
Figure 6: H5–H1 interaction permits the allosteric activation mechanism.

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Acknowledgements

We thank U. F. Lang, C. Chothia, R. Weatheritt, S. Balaji, H. Harbrecht, A. Morgunov, G. Murshudov and N. S. Latysheva for their comments on this work. This work was supported by the Medical Research Council (MC_U105185859; M.M.B., T.F., M.K., A.J.V. and C.N.J.R.; MC_U105197215; C.G.T.), the Swiss National Science Foundation grants 141898, 133810, 31-135754 (D.B.V.), the MRC Centenary Award (A.J.V.), the AFR scholarship from the Luxembourg National Research Fund (C.N.J.R.) and the Boehringer Ingelheim Fond (T.F.). M.M.B. is also a Lister Institute Research Prize Fellow.

Author information

Author notes

  1. A. J. Venkatakrishnan

    Present address: † Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA,

  2. Charles N. J. Ravarani and Dawei Sun: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    Tilman Flock, Charles N. J. Ravarani, A. J. Venkatakrishnan, Melis Kayikci, Christopher G. Tate & M. Madan Babu

  2. Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, 5232, Switzerland

    Dawei Sun & Dmitry B. Veprintsev

  3. Department of Biology, ETH Zurich, Zurich, 8039, Switzerland

    Dawei Sun & Dmitry B. Veprintsev

Authors
  1. Tilman Flock
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Contributions

T.F. collected data, wrote scripts and performed all the analysis. C.N.J.R. helped with data analysis and writing the scripts for structure analysis with T.F. A.J.V. helped T.F. with data analysis. D.S. and D.B.V. contributed experimental results on alanine scanning. M.K. prepared the CGN webserver. C.G.T. helped with aspects of data interpretation. T.F. and M.M.B. designed the project, analysed the results and wrote the manuscript. All authors read and provided their comments on the draft. M.M.B. supervised the project.

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Correspondence to Tilman Flock or M. Madan Babu.

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Extended data figures and tables

Extended Data Figure 1 Human paralogue reference alignment for common Gα numbering system.

a, Reference alignment of all canonical human Gα paralogues. The domain (D), consensus secondary structure (S) and position in the SSE of the human reference alignment (P) are shown on top of the alignment. b, Reference table of the definitions of SSEs used in the CGN nomenclature.

Extended Data Figure 2 Energy estimation of the GPCR–Gα residue contributions and Gα disorder propensity.

a, Energy contribution of single interface residues to the Gαs–β2AR complex calculated with FoldX (T = 298K, pH = 7.0, ionic strength = 0.05M). Conserved Gα residues (blue sequence logo) that were identified to form receptor–Gα inter-protein contacts with conserved GPCR residues (red sequence logo) are shown. The contact network between residues of the β2AR and Gαs is shown (red, conserved receptor residue; blue, conserved Gα residue; grey, variable residues; spheres represent Cα positions and links represent non-covalent contact). b, Consensus disorder plot for all Gα proteins. The mean value of the disorder propensity of all full-length Gα sequences (561 sequences; homologous to all 16 human Gα proteins) is shown as a black line; the standard deviation at each position is shown as light red ribbon. The colour tone of the line indicates the number of gaps at an aligned position (black, no gaps). The left inset shows the disorder propensity of H1. The right inset highlights that H5 is highly structured in its N terminus, and has increased disorder propensity towards the C terminus, which is in agreement with the missing electron density in the 79 structures.

Extended Data Figure 3 Rewiring of consensus contacts between conserved Gα residues upon receptor binding.

CGN numbers and sequence logo for consensus contacts within Gα in the inactive state (left) and GPCR-bound state (right) are shown. Receptor residues are shown in red; H5 residues in dark blue; H1 residues in light blue; and GDP in green. The domains are highlighted with a blue background (G-domain darker blue, H-domain light blue). This figure highlights the most important consensus residue contacts between conserved residues. Additional contacts in the right lobe are discussed in the Supplementary Note and in ref. 25. For a full list of residue contacts, please refer to Supplementary Data.

Extended Data Figure 4 Details of helix H1 linking H5, GDP and the H-domain.

a, This figure expands Fig. 4 from the main text to provide residue-level details of the role of helix H1. Residues forming contacts with H5 are shown in blue, with the H-domain in light blue and with GDP in green. Non-covalent consensus contacts between universally conserved residues at the SSE level (left) and per residue-level (centre). Lines denote non-covalent contacts between residues. The degree of conservation is shown as sequence logo. Residues are numbered according to the CGN. Helix H1 is almost 100% conserved across all 16 Gα types and forms three structural motifs for interactions with H5, the H-domain and GDP (right). b, Average per residue energy contribution to Gα protein stability as calculated from 79 structures from all four Gα subfamilies in the non-receptor-bound signalling states using FoldX (T = 298K, pH = 7.0, ionic strength = 0.05M). The average energy contribution is shown as dots, the standard deviation as bars. c, Per residue detail of Gα-GDP and Gα-GSP (non-hydrolysable GTP analogue) consensus contacts. The bar-plot shows the frequency of finding a contact mediated by topologically equivalent positions with GDP/GSP. Number of side-chain and main-chain contacts are shown as dark grey and light grey bars, respectively. The degree of conservation of contacting residues (calculated from the 561 complete Gα sequences) is represented in the right panel and the consensus sequence for each position is shown.

Extended Data Figure 5 Conserved structural motifs of Gα and known disease and engineered mutations.

a, A universally conserved cluster of π–π and hydrophobic interactions between S2 (PheG.S2.6) and S3 (PheG.S3.3), H1 (MetG.H1.8 and HisG.H1.12) and H5 (PheG.H5.8) links H5 and H1 in the absence of the receptor. Upon receptor binding, residues within this motif (PheG.H5.8 and PheG.S3.3) interact with the conserved Pro and Leu of ICL2 of the receptor as has been shown for Gαs (3sn6) and Gαi (ref. 26). Interrupting the contacts between H5 and H1 seems to be the trigger for transmitting the signal of GPCR binding to helix H1 (which interacts with GDP and the H-domain.) The only conserved residue contact between the H-domain and the G-domain that is not in the hinge region is formed by a universally conserved salt bridge (H-domain ionic latch) between the very N-terminal end of HG of the G-domain (LysG.s5hg.1) and the loop connecting HD and HE in the H domain (AspH.hdhe.5). The hinge region is formed by H1, the loop between H1 and HA, and HF. H1 interacts via (1) a cation–π interaction mediated by a universally conserved residue with the loop connecting H1 and HA (LysG.H1.6 and TyrG.h1ha.4) and (2) a hydrophobic interaction with HF (LysG.H1.9 and LeuH.HF.5). b, Disease and engineered mutations that can be explained by the universal Gα activation mechanism mapped on a Gα protein. Cα position of residues are shown as spheres; mutations at green positions cause spontaneous GDP release by interrupting consensus contacts between conserved residues, thereby ‘mimicking’ the effect of receptor binding to Gα. Pink positions have also been reported to cause disease by constitutively activating Gα. Insertion of an Ala4 or Gly5 after the yellow position separate the H5 transmission and interface module, thereby allowing GPCR binding without triggering GDP release.

Extended Data Figure 6 Helix H5–H1 interaction in Gα provides the allosteric GEF activation mechanism.

a, Schematic representation of structural motifs on H1 that are shared or unique to Gα and Ras. While the part of H1 with the phosphate-binding motif is conserved across both protein families, the C-terminal part is conserved only in Gα. H1 in Gα has three additional residues that allow for extensive residue contacts between H1 and H5. In Ras, these interactions are missing and H5 and H1 are both 3 residues shorter. The consensus sequence and secondary structure of equivalent residues of H1 in Gα and Ras is also depicted. b, Comparison of the residue contact network between topologically equivalent residues in H5 and H1 in the corresponding inactive GDP-bound states of Gα (1got) and Ras (4q21). The weight of the link between SSEs denotes the number of atomic contacts. c, Sequence alignments of H1 and H5 of human Gα and Ras paralogues. The sequence alignment was obtained based on cross-referencing the alignments using the structures of Gα and Ras.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-2, Supplementary Notes and additional references. (PDF 2276 kb)

Supplementary Table 1

This file shows the Gα structure and sequence data. (XLSX 69 kb)

Supplementary Table 2

This file shows the Common Gα reference system data. (XLSX 109 kb)

Supplementary Table 3

This file shows engineered and natural mutations in G proteins reported in literature. (XLSX 52 kb)

Supplementary Data

This zipped file contains 5 files which comprise: Ortholog alignments; Visualization of consensus networks (≧75% consensus score) of the four signaling states with residue conservation represented as B-factor; Visualization of the GPCR-G protein interface in the 3sn6 crystal structure; Gα -Ras alignment; and Consensus residue interaction networks with sequence conservation for Gα and GPCRGα interface residue interaction networks for Gs-β 2-adrenergic receptor and Gt peptides-rhodopsin. (ZIP 3645 kb)

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Flock, T., Ravarani, C., Sun, D. et al. Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173–179 (2015). https://doi.org/10.1038/nature14663

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