CoChat
Sign Up
Cited by 38Receptor Mechanisms and SignalingProtein Kinase Regulation and GTPase SignalingProtein Structure and Dynamics

The DOI matches its Crossref record; 3 of the 3 references it cites were found in a registry or an index.

Mechanistic insights into G-protein coupling with an agonist-bound G-protein-coupled receptor

Open access
Batebi, Hossein, Pérez-Hernández, Guillermo, Rahman, Sabrina N., Lan, Baoliang, Kamprad, Antje, Shi, Mingyu, Speck, David, Tiemann, Johanna K. S., Guixà-González, Ramon, Reinhardt, Franziska, Stadler, Peter F., Papasergi-Scott, Makaía M., Skiniotis, Georgios, Scheerer, Patrick, Kobilka, Brian K., Mathiesen, Jesper M., Liu, Xiangyu, Hildebrand, Peter W.·2024·Nature Structural & Molecular Biology
DOI 10.1038/s41594-024-01334-2PMC PMC12132790

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound G s protein (G s·GDP; hereafter G sGDP) upon coupling to the β 2-adrenergic receptor (β 2 AR) in atomic detail. The binding of G sGDP to the β 2 AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the G s occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this β 2 AR-G sGDP intermediate, G s undergoes an in-plane rotation along the receptor axis to approach the β 2 AR-G sempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1038/s41594-024-01334-2

Cites

3 of 3 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 88

  1. Lefkowitz, R. J. Seven transmembrane receptors: something old, something new. Acta Physiol. 190, 9–19 (2007). · DOI
  2. Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017). · DOI · Paper page
  3. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L. & Khorana, H. G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768–770 (1996). · DOI
  4. Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–557 (2011). · DOI · Paper page
  5. Flock, T. et al. Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173–179 (2015). · DOI
  6. Hauser, A. S. et al. Common coupling map advances GPCR-G protein selectivity. eLife 11, e74107 (2022). · DOI
  7. García-Nafría, J. & Tate, C. G. Structure determination of GPCRs: cryo-EM compared with X-ray crystallography. Biochem. Soc. Trans. 49, 2345 (2021). · DOI
  8. García-Nafría, J. & Tate, C. G. Cryo-EM structures of GPCRs coupled to Gs, Gi and Go. Mol. Cell. Endocrinol. 488, 1–13 (2019). · DOI
  9. Du, Y. et al. Assembly of a GPCR–G protein complex. Cell 177, 1232–1242.e11 (2019). · DOI
  10. Heck, M. & Hofmann, K. P. Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism. J. Biol. Chem. 276, 10000–10009 (2001). · DOI
  11. Scheerer, P. et al. Structural and kinetic modeling of an activating helix switch in the rhodopsin–transducin interface. Proc. Natl Acad. Sci. 106, 10660–10665 (2009). · DOI
  12. Chung, K. Y. et al. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477, 611–615 (2011). · DOI
  13. Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β2AR–G-protein activation. Nature 547, 68–73 (2017). · DOI
  14. Okashah, N. et al. Agonist-induced formation of unproductive receptor-G12 complexes. Proc. Natl Acad. Sci. USA 117, 21723–21730 (2020). · DOI
  15. Liu, X. et al. Structural Insights into the process of GPCR–G protein complex formation. Cell 177, 1243–1251.e12 (2019). · DOI
  16. Mahoney, J. P. & Sunahara, R. K. Mechanistic insights into GPCR–G protein interactions. Curr. Opin. Struct. Biol. 41, 247–254 (2016). · DOI
  17. Jang, W., Lu, S., Wu, G. & Lambert, N. A. The role of G protein confirmation in receptor-G protein selectivity. Nat. Chem. Biol. 19, 687–694 (2023). · DOI
  18. Tsutsumi, N. et al. Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins. Sci. Adv. 8, 5442 (2022). · DOI
  19. Huang, S. K. et al. Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling. Cell 184, 1884–1894.e14 (2021). · DOI
  20. Huang, S. K. et al. Mapping the conformation landscape of the stimulatory heterotrimeric G protein. Nat. Struct. Mol. Biol. 30, 502–511 (2023). · DOI
  21. Sadler, F. et al. Autoregulation of GPCR signalling through the third intracellular loop A FRET-based approach to probe ICL3 conformation. Nature 615, 734–741 (2023). · DOI
  22. Fleetwood, O., Carlsson, J. & Delemotte, L. Identification of ligand-specific G protein-coupled receptor states and prediction of downstream efficacy via data-driven modeling. eLife 10, e60715 (2021). · DOI
  23. Panel, N. et al. Design of drug efficacy guided by free energy simulations of the β2-adrenoceptor. Angew. Chem. Int. Ed. Engl. 62, e202218959 (2023). · DOI
  24. Robinson, G. A., Butcher, R. W. & Sutherland, E. W. Cyclic AMP. Ann. Rev. Biochem. 37, 149–174 (1968). · DOI
  25. Haga, T. et al. Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells. Proc. Natl Acad. Sci. USA 74, 2016–2020 (1977). · DOI
  26. Manglik, A., Kobilka, B. K. & Steyaert, J. Nanobodies to study G protein-coupled receptor structure and function. Annu. Rev. Pharmacol. Toxicol. 57, 19–37 (2017). · DOI
  27. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D. & Bourne, H. R. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature 363, 274–276 (1993). · DOI
  28. Rose, A. S. et al. Position of transmembrane helix 6 determines receptor G protein coupling specificity. J. Am. Chem. Soc. 136, 11244–11247 (2014). · DOI
  29. Selçuk, B. & Adebali, O. Common and selective signal transduction mechanisms of GPCRs. Prog. Mol. Biol. Transl. Sci. 195, 89–100 (2023). · DOI
  30. Kumar, V. et al. GDP release from the open conformation of Gα requires allosteric signaling from the agonist-bound human β2 adrenergic receptor. J. Chem. Inf. Model. 60, 4064–4075 (2020). · DOI
  31. Hofmann, K. P. et al. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci. 34, 540–552 (2009). · DOI · Paper page
  32. Kang, Y. et al. Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558, 553–558 (2018). · DOI
  33. de Mars, G., Fanelli, F. & Puett, D. The extreme C-terminal region of Gαs differentially couples to the luteinizing hormone and β2-adrenergic receptors. Mol. Endocrinol. 25, 1416–1430 (2011). · DOI
  34. Semack, A., Sandhu, M., Malik, R. U., Vaidehi, N. & Sivaramakrishnan, S. Structural elements in the Gαs and Gβq C termini that mediate selective G protein-coupled receptor (GPCR) signaling. J. Biol. Chem. 291, 17929–17940 (2016). · DOI
  35. Hamm, H. E. et al. Site of G protein binding to rhodopsin mapped with synthetic peptides from the α subunit. Science 241, 832–835 (1988). · DOI
  36. Itoh, Y., Cai, K. & Khorana, H. G. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent. Proc. Natl Acad. Sci. USA 98, 4883–4887 (2001). · DOI
  37. Loewen, M. C. et al. Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin. Proc. Natl Acad. Sci. USA 98, 4888–4892 (2001). · DOI
  38. Herrmann, R., Heck, M., Henklein, P., Hofmann, K. P. & Ernst, O. P. Signal transfer from GPCRs to G proteins: role of the Gα N-terminal region in rhodopsin-transducin coupling. J. Biol. Chem. 281, 30234–30241 (2006). · DOI
  39. Natochin, M., Muradov, K. G., McEntaffer, R. L. & Artemyev, N. O. Rhodopsin recognition by mutant Gsα containing C-terminal residues of transducin. J. Biol. Chem. 275, 2669–2675 (2000). · DOI
  40. Blahos, J. et al. Extreme C terminus of G protein α-subunits contains a site that discriminates between Gi-coupled metabotropic glutamate receptors. J. Biol. Chem. 273, 25765–25769 (1998). · DOI
  41. Masuho, I. et al. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors. Sci. Signal. 8, ra123 (2015). · DOI
  42. Schihada, H., Shekhani, R. & Schulte, G. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Sci. Signal. 14, 1653 (2021). · DOI
  43. Landis, C. A. et al. GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 340, 692–696 (1989). · DOI
  44. Wieland, T., Kreiss, J., Gierschik, P. & Jakobs, K. H. Role of GDP in formyl-peptide-receptor-induced activation of guanine-nucleotide-binding proteins in membranes of HL 60 cells. Eur. J. Biochem. 205, 1201–1206 (1992). · DOI
  45. Sun, X., Singh, S., Blumer, K. J. & Bowman, G. R. Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding. eLife 7, e38465 (2018). · DOI
  46. Hu, Q. & Shokat, K. M. Disease-causing mutations in the G protein Gαs subvert the roles of GDP and GTP. Cell 173, 1254–1264.e11 (2018). · DOI
  47. Dror, R. O. et al. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 348, 1361–1365 (2015). · DOI
  48. Karl Pearson, F. R. S. LIII. On lines and planes of closest fit to systems of points in space. Philos. Mag. J. Sci. 2, 559–572 (1901). · DOI
  49. Wrigley, C. & Neuhaus, J. O. The use of an electronic computer in principal axes factor analysis. J. Educ. Psychol. 46, 31–41 (1955). · DOI
  50. Ma, X. et al. Analysis of β2AR–Gs and β2AR–Gi complex formation by NMR spectroscopy. Proc. Natl Acad. Sci. 117, 23096–23105 (2020). · DOI
  51. Draper-Joyce, C. J. et al. Structure of the adenosine-bound human adenosine A1 receptor–Gi complex. Nature 558, 559–563 (2018). · DOI
  52. Koehl, A. et al. Structure of the µ-opioid receptor–Gi protein complex. Nature 558, 547–552 (2018). · DOI
  53. Filipek, S. Molecular switches in GPCRs. Curr. Opin. Struct. Biol. 55, 114–120 (2019). · DOI
  54. Huang, W. et al. Structural insights into µ-opioid receptor activation. Nature 524, 315–321 (2015). · DOI
  55. Papasergi-Scott, M. M. et al. Time-resolved cryo-EM of G-protein activation by a GPCR. Nature https://doi.org/10.1038/s41586-024-07153-1 (2024). · DOI
  56. Zhang, B., Zhang, Y., Shacter, E. & Zheng, Y. Mechanism of the guanine nucleotide exchange reaction of Ras GTPase—evidence for a GTP/GDP displacement model. Biochemistry 44, 2566–2576 (2005). · DOI
  57. Vetter, I. R. & Wittinghofer, A. The guanine nucleotide-binding switch in three dimensions. Science 294, 1299–1304 (2001). · DOI · Paper page
  58. Heng, J. et al. Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor. Nat. Commun. 14, 2005 (2023). · DOI
  59. Rubenstein, R. C., Wong, S. K.-F. & Ross, E. M. The hydrophobic tryptic core of the β-adrenergic receptor retains Gs regulatory activity in response to agonists and thiols. J. Biol. Chem. 262, 16655–16662 (1987). · DOI
  60. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008). · DOI · Paper page
  61. Dror, R. O. et al. Identification of two distinct inactive conformations of the β2-adrenergic receptor reconciles structural and biochemical observations. Proc. Natl Acad. Sci. USA 106, 4689–4694 (2009). · DOI
  62. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983). · DOI
  63. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010). · DOI
  64. Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2016). · DOI
  65. Vanommeslaeghe, K. et al. CHARMM general force field: a force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J. Comput. Chem. 31, 671–690 (2010). · DOI
  66. Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015). · DOI · Paper page
  67. Pérez-Hernández, G. & Hildebrand, P. W. mdciao: accessible analysis and visualization of molecular dynamics simulation data. Preprint at bioRxiv https://doi.org/10.1101/2022.07.15.500163 (2022). · DOI
  68. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996). · DOI · Paper page
  69. Rose, A. S. & Hildebrand, P. W. NGL viewer: a web application for molecular visualization. Nucleic Acids Res. 43, W576 (2015). · DOI · Paper page
  70. Tiemann, J. K. S., Guixà-González, R., Hildebrand, P. W. & Rose, A. S. MDsrv: viewing and sharing molecular dynamics simulations on the web. Nat. Methods 14, 1123–1124 (2017). · DOI
  71. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007). · DOI
  72. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981). · DOI
  73. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993). · DOI
  74. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997). · DOI
  75. McGibbon, R. T. et al. MDTraj: a modern open library for the analysis of molecular dynamics trajectories. Biophys. J. 109, 1528–1532 (2015). · DOI
  76. Scherer, M. K. et al. PyEMMA 2: a software package for estimation, validation, and analysis of Markov models. J. Chem. Theory Comput. 11, 5525–5542 (2015). · DOI
  77. d’Errico, M., Facco, E., Laio, A. & Rodriguez, A. Automatic topography of high-dimensional data sets by non-parametric density peak clustering. Inform. Sci. 560, 476–492 (2021). · DOI
  78. Kästner, J. Umbrella sampling. Wiley Interdiscip. Rev. Comput. Mol. Sci. 1, 932–942 (2011). · DOI
  79. Kumar, S., Rosenberg, J. M., Bouzida, D., Swendsen, R. H. & Kollman, P. A. THE weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem. 13, 1011–1021 (1992). · DOI · Paper page
  80. Isralewitz, B., Gao, M. & Schulten, K. Steered molecular dynamics and mechanical functions of proteins. Curr. Opin. Struct. Biol. 11, 224–230 (2001). · DOI
  81. Jones, E. M. et al. Structural and functional characterization of G protein-coupled receptors with deep mutational scanning. eLife 9, e54895 (2020). · DOI · Paper page
  82. Gaiser, B. I. et al. Probing the existence of a metastable binding site at the β2-adrenergic receptor with homobivalent bitopic ligands. J. Med. Chem. 62, 7806–7839 (2019). · DOI
  83. Hilger, D. et al. Structural insights into differences in G protein activation by family A and family B GPCRs. Science 369, eaba3373 (2020). · DOI
  84. Cahill, T. J. et al. Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc. Natl Acad. Sci. USA 114, 2562–2567 (2017). · DOI
  85. Mathiesen, J. M., Vedel, L. & Bräuner-Osborne, H. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors. Methods Enzymol. 522, 191–207 (2013). · DOI
  86. Pándy-Szekeres, G. et al. GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic Acids Res. 46, D440–D446 (2018). · DOI
  87. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. https://doi.org/10.1016/S1043-9471(05)80049-7 (1995). · DOI
  88. Kooistra, A. J. et al. GPCRdb in 2021: integrating GPCR sequence, structure and function. Nucleic Acids Res. https://doi.org/10.1093/nar/gkaa1080 (2021). · DOI · Paper page

Metadata

Authors
Batebi, H., Pérez-Hernández, G., Rahman, S. N., Lan, B., Kamprad, A., Shi, M., Speck, D., Tiemann, J. K. S., Guixà-González, R., Reinhardt, F., Stadler, P. F., Papasergi-Scott, M. M., Skiniotis, G., Scheerer, P., Kobilka, B. K., Mathiesen, J. M., Liu, X., & Hildebrand, P. W.
Affiliations
  • Freie Universität Berlin (DE)
  • University of Copenhagen (DK)
  • Center for Life Sciences (CN)
  • Humboldt-Universität zu Berlin (DE)
  • Novozymes (Denmark) (DK)
  • Leipzig University (DE)
  • Stanford Medicine (US)
Corresponding author
Hildebrand, Peter W.
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2024-06-12
Volume
31
Issue
11
Pages
1692-1701
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
DOI
10.1038/s41594-024-01334-2
PMC
PMC12132790
OpenAlex
W4399586164
Cited by
38
References
3
Citations by year
2026: 17 · 2025: 16 · 2024: 5
FWCI
5.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • Research Square (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, other-oa)
  • PubMed Central (acceptedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, other-oa)
Funders
  • Philipps-Universität Marburg
  • Santa Fe Institute
  • European Commission (HORIZON2020, 956314)
  • Friedrich-Alexander-Universität Erlangen-Nürnberg (440719683)
  • Deutsche Forschungsgemeinschaft (390540038, EXC 2008/1390540038, subproject A01, EXC 2008/1, SFB1078, CRC 1423, subproject C01, 440719683, CRC 1078, 421152132, 221545957)
  • National Natural Science Foundation of China (32122041)
  • Universidad Nacional de Colombia
  • Universität Wien
  • Tsinghua University
  • Stiftung Charité
  • Berlin Institute of Health
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (K99HL161406)
  • National Institute of Neurological Disorders and Stroke (R01NS028471, R35NS137408)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
G protein coupling, G protein-coupled receptors, Gs protein, GDP release, guanine nucleotide exchange, beta2-adrenergic receptor, allosteric communication, nucleotide binding site, signal transduction
MeSH terms
Allosteric Regulation, Binding Sites, Guanosine Diphosphate, Humans, Protein Binding, Protein Conformation, Signal Transduction, Receptors, Adrenergic, beta-2, GTP-Binding Protein alpha Subunits, Gs, Molecular Dynamics Simulation

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review