Abstract
Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1,2,3,4,5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.
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Acknowledgements
The cryo-EM data were collected at the Advanced Center for Electron Microscopy at the Shanghai Institute of Materia Medica, the Chinese Academy of Sciences (CAS). This work was supported by the CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); the Young Innovator Association of the CAS (2018325 and Y2022078 to L.-H.Z. and 2019282 to K.Wang); the National Natural Science Foundation of China (32071203 to L.-H.Z.; 32130022 and 82121005 to H.E.X.); the National Key R&D Program of China (2019YFA0904200) and the SA-SIBS Scholarship Program to L.-H.Z.; the Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X. and 23ZR1475200 to L.-H.Z.); State Key Laboratory of Drug Research (SKLDR-2023-TT-04).
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L.-H.Z. designed the expression constructs, purified the complexes, prepared the final samples for collection of cryo-EM data relating to the structure, prepared the cryo-EM grids, participated in model building and function assays, carried out structure and function data analysis, prepared figures and wrote the manuscript; W.H. and K.Wu collected the cryo-EM data, Q.Y. and J.L. carried out map calculations, Q.Y. and H.S. built and refined the structure models; X.H. carried out structure modelling and volume calculation; L.-H.Z., Q.H., Y.G. and Y.L. constructed functional plasmids, Q.H. carried out signalling experiments under the supervision of L.-H.Z.; K.Wang. and J.S. supplied materials; L.-H.Z. and H.E.X. conceived the project and wrote the manuscript.
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H.E.X. is a founder of Cascade Pharmaceuticals. All other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Confocal microscopy of HEK-293 cells expressing PTH1R with β-arrestin-mcherry.
(a–b) The cells expressing GFP-PTH1R (green) with β-arrestin-mcherry (red) and treated with PTH or PCO371 (5 min) were shown with significant recruitment of both β-arrestin 1 and β-arrestin 2 to the cells membranes after PTH stimulation (top row), whereas PCO371 and vehicle control did not stimulate membrane association of both β-arrestin1 and β-arrestin 2 (middle and bottom row). (c–d) HEK-293 cells coexpressing untagged PTH1R and β-arrestin -GFP and treated with PTH (5 min) had significant recruitment of both β-arrestin 1 and β-arrestin 2 to cell membranes (top row). While β-arrestin-GFP (green) appeared diffuse in cells treated with PCO371 stimulation and untreated control (vehicle) (middle and bottom row). Data are from three independent experiments (n = 3). Scale bar, 10 μm.
Extended Data Fig. 2 Construct of receptor and purification of the PCO371-PTH1R-Gs complex.
(a) Snake plot diagram of the PTH1R-LgBiT construct. (b) The size-exclusion chromatography elution profile on Superdex200 Increase 10/300GL (left panel) and SDS-PAGE analysis (right panel) of the PCO371-PTH1R-Gs complex.
Extended Data Fig. 3 Single particle cryo-EM data analysis of the PCO371-PTH1R-Gs complex.
(a) Representative cryo-EM micrograph from 5364 movies of the PCO371-PTH1R-Gs complex and representative 2D class averages with distinct secondary structure features from different views. (b) Data processing flowchart of PCO371-PTH1R-Gs complex by CryoSPARC and Relion. (c) Color cryo-EM map of the PCO371-PTH1R-Gs complex, showing local resolution (Å) calculated using Relion. (d) “Gold-standard” FSC curve of the PCO371-PTH1R-Gs complex, with the global resolution defined at the FSC = 0.143 is 2.57 Å.
Extended Data Fig. 4 Cryo-EM density maps of the PCO371-PTH1R-Gs protein structures.
Cryo-EM density map and the model of the PCO371-PTH1R-Gs structure are shown for all transmembrane helices and helix 8 of PTH1R, PCO371, and Gαs-α5 helix. The model is shown in stick representation. All of them display good density.
Extended Data Fig. 5 Structure comparisons of the active state and inactive state of PTH1R induced by different ligands.
(a–d) Structure comparisons are between the active state complexes with PTH and with PCO371. Superimposition of PTH1R from PDB: 8HA0 (PTH1R: royal blue, PTH: light coral) and the PCO371-bound PTH1R structure (PTH1R: light sea green, PCO371: crimson) reveals different peptide- and PCO371-binding sites. (a–b) Side view of different binding pockets and conformational changes in receptors; (c) Extracellular view and (d) intracellular view of PTH1R conformational changes. (e–h) Superimposed structures of PCO371-bound PTH1R in the active state, and ePTH-bound PTH1R in the inactive state from PDB: 6FJ3 (PTH1R: dark gray, ePTH: dark khaki) and the PCO371-bound PTH1R structure (PTH1R: light sea green, PCO371: crimson). (e–f) Side view of different binding pockets and conformational changes in receptors; (g) Extracellular view and (h) intracellular view of PTH1R conformational changes.
Extended Data Fig. 6 Comparisons of peptide hormone binding pockets and small molecule agonist binding sites of class B GPCRs.
The PCO371 binding pocket is different from both peptide binding pockets and other small molecule binding pockets of class B GPCRs. (a–d) Comparisons the ligand-binding pockets between PCO371 and peptides of PTH1R. The receptor is shown in surface representation and colored in light sea green and PCO371 in crimson is shown as sticks. In three PTH-, PTHrP- and LA-PTH-bound PTH1R-Gs complex structures, the receptors are shown in surface representation and colored in royal blue, dark khaki and dark orchid, respectively. PTH, PTHrP and LA-PTH are colored in light coral, medium violet red and forest green, respectively. They are shown as sticks and ribbon (PDB: 8HA0, 8HAF and 6NBF). G proteins and Nb35 are omitted for clarity. (e–l) Comparisons of small molecule agonist binding sites of class B GPCRs. Superimposition of the PTH1R (light sea green) in complex with Gs bound to PCO371 (crimson) with the GLP1R in complexes with Gs bound to different non-peptidic ligands, including small molecule agonists: TT-OAD2(PDB: 6ORV; TT-OAD2: dark orchid, GLP1R: burly wood); RGT1383 (PDB: 7C2E; RGT1383: green, GLP1R: light salmon); PF-06882961(PDB: 6X1A; PF-06882961: dark orange, GLP1R: thistle); CHU-128 (PDB: 6X19; CHU-128: yellow, GLP1R: rosy brown); LY3502970 (PDB: 6XOX; LY3502970: sandy brown, GLP1R: silver); Boc5 (PDB: 7X8R; Boc5:blue, GLP1R: dark sea green) and WB4-24 (PDB:7X8S; WB4-24: indigo, GLP1R: light steel blue) and with an allosteric ligand, Compound 2, (PDB: 7EVM; Compound 2: goldenrod, GLP1R: tomato), G proteins and Nb35 are omitted for clarity.
Extended Data Fig. 7 Chemical structure of PCO371 and PCO371-mediated cAMP production by receptors containing alanine mutants of key residues in PCO371 binding pocket.
(a) The chemical structure of PCO371 is comprised of the head imidazolidinone, the middle dimethylphenyl, the sulfonamide linker, the piperidine motif, the middle spiro-imidazolone, and the tail trifluoromethoxy phenyl. (b) PCO371-mediated cAMP production by receptors containing alanine mutants of key residues within TM2, TM3, TM6, TM7 and H8. Data from three independent experiments (n = 3) performed in technical triplicate are presented as mean ± s.e.m.
Extended Data Fig. 8 The similarity and the difference of PTH1R in G protein-coupling by hormone peptide and small molecule agonist.
(a) Structural comparison of G protein in different ligands bound PTH1R-Gs complex structures. (b) Close up of the αN and Gαs-α5 helix of Gαs, which form interactions with ICL2 and TMD helix bundles in all G protein bound complex structures, showing similar G protein conformation, but the noteworthy difference is that the C-terminal of Gαs-α5 helix makes additional interactions with the small molecule agonist. (c) Good cryo-EM density supports ligand interact with Gαs. (d-f) The similar set of interactions between the C-terminal of Gαs-α5 helix with the receptor. E392 shifts outward due to steric clash. Y391, E392, and L393 form additional interactions with PCO371.
Extended Data Fig. 9 Key residues for PCO371 selectivity among class B GPCRs.
(a) Structural comparison of receptors and ligands among PCO371-PTH1R-Gs and TT-OAD2-GLP1R-Gs and TIP39-PTH1R-Gs complexes. (a-b) Structural comparison of the cytoplasmic regions of PTH1R, PTH2R and GLP1R during the receptor activation. (c) Structural comparison of P4156.47b and L3706.47b in PTH receptors. (d-e) Different conformations of residues in the active PTH1R, and GLP1R that are involved the interface of PCO371 in receptor activation. (f) Stimulation of cAMP production by PCO371 in the WT and mutants of GLP1R. Data from three independent experiments (n = 3) performed in technical triplicate are presented as mean ± s.e.m. (g) Stimulation of cAMP production by the cognate ligands of PTH1R, PTH2R and GLP1R in mutants of receptors. Data from three independent experiments (n = 3) performed in technical triplicate are presented as mean ± s.e.m.
Supplementary information
Supplementary Fig. 1
Raw SDS–polyacrylamide gel electrophoresis images of the PCO371–PTH1R–Gs complex.
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Zhao, LH., He, Q., Yuan, Q. et al. Conserved class B GPCR activation by a biased intracellular agonist. Nature 621, 635–641 (2023). https://doi.org/10.1038/s41586-023-06467-w
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DOI: https://doi.org/10.1038/s41586-023-06467-w
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