Abstract
G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR–G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein–receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.
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Data availability
The atomic coordinates of β2AR–GsEMPTY (frames 1–20) have been deposited in the Protein Data Bank (PDB) under accession codes 8GDZ, 8GE1, 8GE2, 8GE3, 8GE4, 8GE5, 8GE6, 8GE7, 8GE8, 8GE9, 8GEA, 8GEB, 8GEC, 8GED, 8GEE, 8GEF, 8GEG, 8GEH, 8GEI and 8GEJ, respectively. The atomic coordinates of β2AR–GsGTP(Merged) (Frames 1–20) have been deposited in the PDB under accession codes 8GFV, 8GFW, 8GFX, 8GFY, 8GFZ,8GG0, 8GG1, 8GG2, 8GG3, 8GG4, 8GG5, 8GG6, 8GG7, 8GG8, 8GG9, 8GGA, 8GGB, 8GGC, 8GGE and 8GGF, respectively; along with the coordinates from corresponding localized maps of β2AR under accession codes 8GGI, 8GGJ, 8GGK, 8GGL, 8GGM, 8GGN, 8GGO, 8GGP,8GGQ, 8GGR, 8GGS, 8GGT, 8GGU, 8GGV, 8GGW, 8GGX, 8GGY, 8GGZ, 8GH0 and 8GH1, respectively. The atomic coordinates of β2AR–GsGTP(Merged) (classes A–T) have been deposited in the PDB under accession codes 8UNL, 8UNM, 8UNN, 8UNO, 8UNP, 8UNQ, 8UNR, 8UNS, 8UNT, 8UNU, 8UNV, 8UNW, 8UNX, 8UNY, 8UNZ, 8UO0, 8UO1, 8UO2, 8UO3 and 8UO4, respectively. Cryo-EM maps of β2AR–GsEMPTY (frames 1–20) have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-29951, EMD-29952, EMD-29953, EMD-29954, EMD-29955, EMD-29956, EMD-29958, EMD-29959, EMD-29960, EMD-29961, EMD-29962, EMD-29964, EMD-29965, EMD-29966, EMD-29967, EMD-29968, EMD-29969, EMD-29970, EMD-29971 and EMD-29972, respectively. Cryo-EM maps of β2AR–GsGTP(5sec) (frames 1–20) have been deposited in the EMDB under accession codes EMD-40096, EMD-40097, EMD-40098, EMD-40099, EMD-40100, EMD-40101, EMD-40102, EMD-40103, EMD-40104, EMD-40105, EMD-40106, EMD-40107, EMD-40108, EMD-40109, EMD-40110, EMD-40111, EMD-40112, EMD-40113, EMD-40114 and EMD-40115, respectively. Cryo-EM maps of β2AR–GsGTP(10sec) (frames 1–20) have been deposited in the EMDB under accession codes EMD-40116, EMD-40117, EMD-40118, EMD-40119, EMD-40120, EMD-40121, EMD-40122, EMD-40123, EMD-40124, EMD-40125, EMD-40126, EMD-40127, EMD-40128, EMD-40129, EMD-40130, EMD-40131, EMD-40132, EMD-40133, EMD-40134 and EMD-40135, respectively. Cryo-EM maps of β2AR–GsGTP(17sec) (frames 1–20) have been deposited in the EMDB under accession codes EMD-40136, EMD-40137, EMD-40138, EMD-40139, EMD-40140, EMD-40141, EMD-40142, EMD-40143, EMD-40144, EMD-40145, EMD-40146, EMD-40147, EMD-40148, EMD-40149, EMD-40150, EMD-40151, EMD-40152, EMD-40153, EMD-40154 and EMD-40155, respectively. Cryo-EM maps of β2AR–GsGTP(Merged) (frames 1–20) have been deposited in the EMDB under accession codes EMD-29985, EMD-29986, EMD-29987, EMD-29988, EMD-29989, EMD-29990, EMD-29991, EMD-29992, EMD-29993, EMD-29994, EMD-29995, EMD-29996, EMD-29997, EMD-29998, EMD-29999, EMD-40000, EMD-40001, EMD-40002, EMD-40004 and EMD-40005, respectively, along with the corresponding localized maps of β2AR under accession codes EMD-40009, EMD-40010, EMD-40011, EMD-40012, EMD-40013, EMD-40014, EMD-40015, EMD-40016, EMD-40017, EMD-40018, EMD-40019, EMD-40020, EMD-40021, EMD-40022, EMD-40023, EMD-40024, EMD-40025, EMD-40026, EMD-40027 and EMD-40028, respectively; and localized G-protein maps under accession codes EMD-40156, EMD-40157, EMD-40158, EMD-40159, EMD-40160, EMD-40161, EMD-40163, EMD-40164, EMD-40165, EMD-40166, EMD-40167, EMD-40168, EMD-40169, EMD-40170, EMD-40171, EMD-40172, EMD-40173, EMD-40174, EMD-40175 and EMD-40176, respectively. Cryo-EM maps of β2AR–GsGTP(Merged) (classes A–T) have been deposited in the EMDB under accession codes EMD-42408, EMD-42409, EMD-42410, EMD-42411, EMD-42412, EMD-42413, EMD-42414, EMD-42415, EMD-42416, EMD-42417, EMD-42418, EMD-42419, EMD-42420, EMD-42421, EMD-42422, EMD-42423, EMD-42424, EMD-42425, EMD-42426 and EMD-42427, respectively. Raw cryo-EM image data have been deposited in the Electron Microscopy Public Image Archive (EMPIAR) under ascension codes EMPIAR-11855, EMPIAR-11856, EMPIAR-11857 and EMPIAR-11858 for the β2AR–GsEMPTY, β2AR–GsGTP(5sec), β2AR–GsGTP(10sec) and β2AR–GsGTP(17sec) datasets, respectively. Visualizations of MD trajectories are made available via MDsrv sessions included in a Zenodo dataset associated with this manuscript (https://doi.org/10.5281/zenodo.10548787)93. Coordinates of comparison structures were available and obtained through the Protein Data Bank, under accession codes: 3SN6 (ref. 2), 1AZT (ref. 45), 2RH1 (ref. 47), 7L0Q (ref. 30) and 7RKF (ref. 50).
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Acknowledgements
Research reported in this publication was supported by equipment access through the Stanford Cryo-Electron Microscopy Center (cEMc). This work was funded by National Institutes of Health grants K99HL16140601 (to M.M.P.-S.), R01GM083118 (to G.S. and B.K.K.) and R01NS028471 (to B.K.K.) and Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) DFG grants GRK 1910 and GM 13/14-1 (to P.G.) and SFB1423, project number 421152132, subproject C01, Stiftung Charité and the Einstein Center Digital for Future (to P.W.H.). We acknowledge the scientific support and HPC resources provided by the Erlangen National High Performance Computing Center (NHR@FAU) of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) under NHR project p101ae. NHR funding is provided by federal and Bavarian state authorities. NHR@FAU hardware is partially funded by the German Research Foundation (DFG)-440719683.
Author information
Authors and Affiliations
Contributions
M.M.P.-S. prepared cryo-EM grids, collected, analysed and processed cryo-EM data to generate final cryo-EM reconstructions, built and refined atomic models, collected and processed negative-stain EM data, analysed data, prepared figures and wrote the manuscript. G.P.-H. performed data analysis of cryo-EM models and MD simulations and contributed to figure development. H.B. performed MD simulations and data analysis and contributed to figure development. Y.G. prepared complex and prepared cryo-EM grids and generated a preliminary cryo-EM reconstruction for the 5 s GTP time point. G.E. prepared cryo-EM grids, collected, analysed and generated preliminary reconstructions for the 3D-classified nucleotide-free states with the assistance of A.B.S. G.E. and D.H. optimized conditions to obtain stable complexes for the study. D.H. purified and prepared β2AR–Gs complexes. O.P. collected cryo-EM data for the 5 s GTP time point. M.C. purified β2AR and Gs, and prepared β2AR–Gs complexes. F.H. purified Gs and assisted complex preparation. L.M. synthesized c-Epi. P.G. supervised the synthesis of c-Epi. B.K.K. oversaw protein purification and β2AR–Gs complexation. P.W.H. supervised molecular dynamics studies. G.S. oversaw cryo-EM studies and conceived and supervised the project. M.M.P.-S. and G.S. wrote the manuscript.
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G.S. is a co-founder of, and consultant for, Deep Apple Therapeutics. B.K.K. is a co-founder of, and consultant for, ConfometRx.
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Extended data figures and tables
Extended Data Fig. 1 Cryo-EM processing and reconstruction of β2AR-GsEMPTY.
a, Flow chart outlining the cryo-EM processing of β2AR-GsEMPTY complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian filtered map outline to show micelle and AHD densities. b, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 2 Dynamic residency of Gα AHD in open and closed positions.
a, Measurement of the real time of vitrification using a Vitrobot. The Vitrobot timing is the sum of user programmed blot time and wait time, 2 sec (4.95 sec ± 0.026 S.E.M., n = 10), 7 sec (9.99 sec ± 0.029 S.E.M., n = 10), 14 sec (17.02 sec ± 0.040 S.E.M., n = 10), where n indicates number of measurements recorded. Individual data points shown. b-h, To determine the residency of the AHD between open and closed positions in cryo-EM reconstructions, the AHD was docked into frames 1 (maximally open AHD) and 20 (maximally closed AHD) of each 3DVA trajectory (c-d, f-h) or 3D classes ordered from left, class A, to right, class T, by percent contribution of particles from the 17 sec dataset (e), a region of 6 Å from the docked structures was used to define ‘fully open’ or ‘fully closed’ respectively, b, and the volume of cryo-EM map at a threshold level of 0.05 that was enclosed in the defined regions was determined, c-g. i, Location of Gα AHD in relation to Gβ. The crystal structure (PDB:3SN6) locates the Gα AHD (grey) adjacent to Gβ blades 1 (red) and 2 (orange) and interacting with blade 2. In contrast, the location of the cryo-EM density that corresponds to the AHD lies adjacent to Gβ blades 2 and 3 (yellow) in both the nucleotide-free and GTP conditions. The cryo-EM structure of NTSR1-Gi also has an open AHD adjacent to blades 2 and 3, but in a different orientation. Structures have been aligned to Gβ. In the middle panels, the cryo-EM density envelope (Gaussian filtered, σ = 2) of the unsharpened map is shown with the density corresponding to the location of the AHD shaded in grey.
Extended Data Fig. 3 Cryo-EM processing and reconstruction of β2AR-GsGTP(5sec).
a, Flow chart outlining the cryo-EM processing of β2AR-GsGTP(5sec) complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian-filtered map outline to show micelle and AHD densities. b, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 4 Cryo-EM processing and reconstruction of β2AR-GsGTP(10sec).
a, Flow chart outlining the cryo-EM processing of β2AR-GsGTP(10sec) complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian filtered map outline to show micelle and AHD densities. b, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 5 Cryo-EM processing and reconstruction of β2AR-GsGTP(17sec).
a, Flow chart outlining the cryo-EM processing of β2AR-GsGTP(17sec) complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian filtered map outline to show micelle and AHD densities. b, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 2 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 6 Cryo-EM processing and reconstruction of β2AR-GsGTP(Merged).
a, Flow chart outlining the cryo-EM processing of β2AR-GsGTP(Merged) complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian filtered map outline to show micelle and AHD densities. The percent contribution of particles from each dataset to each local refinement is shown next to each reconstruction (orange, 5 sec.; blue, 10 sec.; green, 17 sec.). b, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 7 Cryo-EM processing and reconstruction of β2AR-GsGTP(Merged) 3D classes.
a, Continuation of the flow chart in Extended Data Fig. 6 outlining the cryo-EM processing of β2AR-GsGTP(Merged) complex using cryoSPARC29,65. Local refinement reconstructions are shown with a Gaussian filtered map outline to show micelle and AHD densities. The percent contribution of particles from each dataset to each local refinement is shown next to each reconstruction (orange, 5 sec.; blue, 10 sec.; green, 17 sec.). b, Local resolution of projections used in final cryo-EM reconstructions arising from 3D classification of particles without alignment. See Supplementary Fig. 1 for associated 3DFSC94 curves, directional orientation, power spectra, and angular distribution maps; and see Supplementary Table 2 for a table of sphericity scores.
Extended Data Fig. 8 GTP-bound Gαs in the β2AR-Gs complex transitions to a similar structure as activated Gαs-GTPγS.
a-g, Structures comparing the overall architecture of the first and last frames of the β2AR-GsEMPTY and β2AR-GsGTP trajectories with ‘checkpoint’ crystal structures of nucleotide free β2AR-Gs complex PDB:3SN6 and activated Gαs-GTPγS. Models are aligned to the RHD. h, Rotation of Gs in relation to receptor (aligned) over structures of β2AR-GsGTP cryo-EM structural transition frames. i, Placement of α5 Phe in relation to hydrophobic pocket on RHD β-sheets. Rendering style inspired by Jang et al.19. The residue F376 of Frame 20 ( + GTP condition), in the bottom-middle panel, is translucent blue to indicate it has been built in as a likely position but is stubbed in our deposited molecular model of that frame. j-k, The transition state of US28-G11GDP captured in the process of nucleotide release is similar to that of β2AR-GsGTP (frame 20). l-m, Trace of the root-mean-square-deviation (RMSD) over the 20 β2AR-GsGTP structural transition frames. Structures have been aligned to the rigid elements of the Gαs-RHD, and the RMSD has been computed both for the Cα atoms of the whole Gαs-RHD (l) and just of the α5 helix (m). The traces show that for both the Gαs-RHD as a whole and the α5 helix, the early frames are structurally closer to PDB:3SN6, whereas the last three frames, from 18 onwards, are closer to PDB:1AZT.
Extended Data Fig. 9 Local refinement of β2AR-GsGTP(Merged).
a, 2D class averages arising from the 47,951 particles contributing to frame 20 of the β2AR-GsGTP(Merged) reconstruction sorted into 100 2D classes. All classes appear to have intact receptor micelle and G protein in the complex. b, Focused cryo-EM reconstructions of β2AR receptor. c, Local resolution of projections used in final cryo-EM reconstructions. See Supplementary Fig. 1 for associated angular distribution maps.
Extended Data Fig. 10 Molecular Dynamics simulations of β2AR-GsGTP intermediate structures.
a Weakened interactions of β2AR and Gs in simulations seeded by later cryo-EM intermediate structures. Chord diagrams show interactions between receptor regions (purple) with Gα regions (gold) coarse-grained to domain segments. Interactions are defined as residue pairs having at least one pair of heavy atoms less than 4 Å apart. Each chord diagram is generated using all the data from triplicate 3 μsec MD trajectories for each seed/condition. The average sum of total contacts for each triplicate #16–20 are 41.6, 35.4, 30.6, 28.2, and 20.6, respectively. b-g, Quantification of movement of TM5 (b, c) and TM6 (d, e) on the extracellular and intracellular sides of β2AR; of the ionic lock with contact frequencies at 4 Å shown inset (f), and of c-Epi ligand (g). Dashed vertical lines represent values of seed structures. h, Sampling of ligand poses over the MD trajectories shown both as discrete transitions between poses (color-coded time traces, see adjacent ligand pose key below panel), as well as in terms of RMSD to the initial pose (solid black line). i, Principal component analysis of the sampled ligand poses, with the positions of selected representative poses superimposed as color-coded circled numbers. j, Superimposition of selected ligand poses shown in ‘i’, showing coverage of the entire ligand binding pocket volume shaded in light purple. k, Representative models of selected ligand pose clusters. TM6 shown in solid purple, c-Epi ligand in orange, transparent lilac colored cloud represents the volume sampled by the ligand across all MD trajectories. The extracellular half of TM7 is hidden to show ligand binding pocket. See also Supplementary Table 6 for detailed population information of ligand poses.
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Papasergi-Scott, M.M., Pérez-Hernández, G., Batebi, H. et al. Time-resolved cryo-EM of G-protein activation by a GPCR. Nature (2024). https://doi.org/10.1038/s41586-024-07153-1
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DOI: https://doi.org/10.1038/s41586-024-07153-1
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