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A method for structure determination of GPCRs in various states

Nature Chemical Biology volume 20, pages 74–82 (2024)Cite this article

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Abstract

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the β2-adrenergic receptor (β2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For β2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.

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Fig. 1: Design strategy of the universal method.
Fig. 2: Characterization of the 1B3 Fab and design of the glue molecule.
Fig. 3: Ligand binding activity and antagonist-bound structure of β2AR.
Fig. 4: Agonist-bound structures of β2AR.
Fig. 5: Conformational changes in ADGRL3 activation.

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Data availability

The three-dimensional cryo-EM density maps of alprenolol–β2AR–mBRIL, formoterol–β2AR–mBRIL, olodaterol–β2AR–mBRIL and ADGRL3–mBRIL structures have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-36361, EMD-36342, EMD-36360 and EMD-36426, respectively. Atomic coordinates for the atomic models of BRIL–1B3, alprenolol–β2AR–mBRIL, formoterol–β2AR–mBRIL, olodaterol–β2AR–mBRIL and ADGRL3–mBRIL structures have been deposited in the PDB under accession numbers 8J7E, 8JJO, 8JJ8, 8JJL and 8JMT, respectively. All relevant data in this paper are included in the paper or the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

Cryo-EM data were collected at the Center of Cryo-Electron Microscopy, University of Science and Technology of China. We thank staff members at the Center for Integrative Imaging, Hefei National Laboratory for Physical Sciences at the Microscale and University of Science and Technology of China for cryo-EM sample examination. We also thank staff members at the Shanghai Synchrotron Radiation Facility for assistance in data collection. This work was supported by the National Key Research and Development Program of China grants 2022YFF1203100 and 2018YFA0902700 and Center for Advanced Interdisciplinary Science and Biomedicine of IHM grant QYZD20220006 (to Y.T.).

Author information

Author notes

  1. These authors contributed equally: Qiong Guo, Binbin He, Yixuan Zhong, Haizhan Jiao.

Authors and Affiliations

  1. Department of Laboratory Medicine, The First Affiliated Hospital of USTC, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, Hefei National Center for Cross-Disciplinary Sciences, Biomedical Sciences and Health Laboratory of Anhui Province, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Qiong Guo, Binbin He, Yixuan Zhong, Qinggong Wang, Qiangqiang Ge, Yongxiang Gao & Yuyong Tao

  2. Kobilka Institute of Innovative Drug Discovery, School of Medicine, The Chinese University of Hong Kong, Shenzhen, China

    Haizhan Jiao, Yang Du & Hongli Hu

  3. Beijing Frontier Research Center for Biological Structure, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Center for Life Sciences, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Yinhang Ren & Xiangyu Liu

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Contributions

Q. Guo and Y.Z. performed antibody selection, characterization and structure determination. Q. Guo, B.H., Y.Z., Y.R. and Y.G. performed the biochemical studies, prepared the cryo-EM samples and collected the cryo-EM data. Q. Guo, Y.Z. and H.J. processed the cryo-EM data. Q.W. and Q. Ge helped with data processing. Y.D. and H.H. supervised cryo-EM data processing. X.L. supervised radioligand binding assays. Y.T. conceived and supervised the project and wrote the paper.

Corresponding authors

Correspondence to Hongli Hu or Yuyong Tao.

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Competing interests

Y.T., Q. Guo, B.H. and Y.Z. have filed an invention patent for the method described in this work. The other authors declare no competing interests.

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Nature Chemical Biology thanks Shoji Maeda, H. Eric Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 GPCR fusion with BRIL.

a, Examples of the crystal structures of GPCRs with fusion partners. Every fusion protein is connected to GPCRs with at least one coil (indicated with a red arrow) except BRIL. b, Representative 2D classifications indicate the heterogeneous conformations in the β2AR-BRIL Fab complex. c, The stability test revealed that β2AR-mBRIL has higher stability than β2AR-BRIL. The experiment was repeated twice independently with similar results. d, Steric clashes between the 5-HT2B receptor (blue, PDB ID: 4ib4) and apo BRIL (gray, PDB ID: 5ym7) loop. e, Examples of the crystal structures of GPCR-BRIL fusion proteins. The linker between BRIL helix II and helix III is not visible in these structures.

Source data

Extended Data Fig. 2 GPCR fusion with mBRIL and ALFA.

a, The replaced loop of BRIL avoids the conflict between BRIL and GPCR. Modeled with 5-HT2B-BRIL (blue, PDB ID: 4ib4). b, Structure alignment of BRIL Fabs and BRIL-GPCR (PDB ID: 4ib4). Both BAG2 and SRP2070Fab are positioned in an inappropriate orientation. c, Equivalent expression levels of β2AR-H8-ALFA fusion proteins. ALFA was fused with different spacers after H8 (left). An eGFP was fused at the C-terminus of the fusion protein for fluorescence-detection size-exclusion chromatography (left). Example of the pull-down result of β2AR-mBRIL-3aa-ALFA with NbALFA (right). The experiment was repeated twice independently with similar results.

Source data

Extended Data Fig. 3 Selection and characterization of the anti-BRIL Fabs.

a, Modeling of a desired BRIL Fab (cyan/green) onto GPCR-BRIL. The BRIL (magenta)-fused 5-HT2B receptor (blue) was used (PDB ID: 4ib4). A scaffold Fab was placed in a favorite position. b, The desired epitope on BRIL. Key residues from the epitope (highlighted as yellow sticks) were mutated into alanines for the ‘counter’ antigen. c, Single colony phage ELISA of the 12 different colonies showed high binding signals to WT BRIL but weak binding signals to mutant BRIL. Data shown are means ± s.e.m. from N = 3 independent experiments performed in technical duplicate. d, The binding affinity of the candidate Fabs for BRIL. The affinity of the 12 antibodies was determined using protein-based ELISA. Seven of them showed high affinity for WT BRIL (left), and all seven Fabs showed low affinity for the mutant BRIL (right). Data shown are means ± s.e.m. from N = 3 independent experiments performed in technical duplicate. e, The binding affinity of the seven Fabs for mBRIL. The affinity was determined by protein-based ELISA and was comparable to that measured with WT BRIL. Data shown are means ± s.e.m. from N = 3 independent experiments performed in technical duplicate. f, Details of the interaction between BRIL and the 1B3 Fab. BRIL is shown in magenta, and the heavy and light chains of Fab are colored green and cyan, respectively. The 1B3 light chain (above) and heavy chain (down) mediated contacts with BRIL. g, The docked model of 1B3 Fab (green for light chain, cyan for heavy chain) and the associated NbFab (orange) on GPCR-BRIL. The structure of the BRIL (magenta)-fused 5-HT2B receptor (blue) was used (PDB ID: 4ib4).

Source data

Extended Data Fig. 4 Design and characterization of the bivalent glue molecule.

a, The predicted binding position of NbALFA (pink). ALFA was fused with different spacers (indicated by the underlined residues). NbALFA was aligned onto the no spacer (left), a one-residue spacer (middle), and a two-residue spacer ALFA models. Three residues normally perform a helical turn. b, The binding ability of the bivalent glue molecule to β2AR-mBRIL and 1B3 Fab. The glue molecule forms a 1:1 complex with β2AR-mBRIL (left) and 1B3 (right) in pull-down assays. The experiment was repeated at least four times independently with similar results.

Source data

Extended Data Fig. 5 Cryo-EM processing and 3D reconstruction workflow for the alprenolol-β2AR-mBRIL complex.

a, Cryo-EM data processing workflow. b, Gold-standard FSC curves of the 3D reconstructions. c, Local resolution map of the complex. d, Cryo-EM density maps and models of the seven transmembrane helices (TM1-7) of the complex. Maps are shown in cyan.

Extended Data Fig. 6 Cryo-EM processing and 3D reconstruction workflow for the formoterol-β2AR-mBRIL complex.

a, Cryo-EM data processing workflow. b, Gold-standard FSC curves of the 3D reconstructions. c, Local resolution map of the complex. d, Cryo-EM density maps and models of the seven transmembrane helices (TM1-7) of the complex. Maps are shown in green.

Extended Data Fig. 7 Cryo-EM processing and 3D reconstruction workflow for the olodaterol-β2AR-mBRIL complex.

a, Cryo-EM data processing workflow. b, Gold-standard FSC curves of the 3D reconstructions. c, Local resolution map of the complex. d, Cryo-EM density maps and models of the seven transmembrane helices (TM1-7) of the complex. Maps are shown in pink.

Extended Data Fig. 8 Optimization of the fusion site and glue molecule for ADGRL3.

a, The predicted model of ADGRL3-mBRIL. NbALFA and 1B3/NbFab were docked through structural alignment. E3/K3 was manually docked. ALFA was fused with a two-residue spacer after H8 (LRTH). b, The detailed design of the glue molecule combination. c, Representative 2D classifications indicate the ‘4 + 5’ combination resulting in more desired particles. A particle with three parts outside the micelle satisfies the design scheme.

Extended Data Fig. 9 Cryo-EM processing and 3D reconstruction workflow for the ADGRL3-mBRIL complex.

a, Cryo-EM data processing workflow. b, Gold-standard FSC curves of the 3D reconstructions. c, Local resolution map of the complex. d, Cryo-EM density maps and models of the seven transmembrane helices (TM1-7) of the complex. Maps are shown in blue.

Extended Data Fig. 10 Structural comparison of inactive ADGRL3 with class B1 members.

Structural comparison of inactive ADGRL3 with inactive glucagon receptor (PDB ID: 5EE7) (a), glucagon-like peptide-1 receptor (PDB ID: 5VEW) (b) and corticotropin-releasing factor receptor 1 (PDB ID: 4Z9G) (c).

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Figs. 1 and 2.

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data for Fig. 1c.

Source Data Fig. 2

Unprocessed gel image of Fig. 2a.

Source Data Fig. 3

Statistical source data for Fig. 3a.

Source Data Extended Data Fig. 1

Unprocessed gel image of Extended Data Fig. 1c.

Source Data Extended Data Fig. 2

Unprocessed gel image of Extended Data Fig. 2c.

Source Data Extended Data Fig. 3

Statistical source data for Extended Data Fig. 3c–e.

Source Data Extended Data Fig. 4

Unprocessed gel image of Extended Data Fig. 4b.

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Guo, Q., He, B., Zhong, Y. et al. A method for structure determination of GPCRs in various states. Nat Chem Biol 20, 74–82 (2024). https://doi.org/10.1038/s41589-023-01389-0

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