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Structures of the human cholecystokinin receptors bound to agonists and antagonists

Nature Chemical Biology volume 17, pages 1230–1237 (2021)Cite this article

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Abstract

Cholecystokinin receptors, CCKAR and CCKBR, are important neurointestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here, we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR–gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

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Fig. 1: Overall structures of the CCKAR and CCKBR.
Fig. 2: Small-molecule ligand-binding pocket of CCKRs.
Fig. 3: Comparison of peptide binding in CCKRs.
Fig. 4: Activation process of CCKAR.

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Data availability

Atomic coordinates for the structures of CCKAR–lintitript, CCKAR–devazepide and CCKAR–NN9056 have been deposited in the RCSB PDB under accession codes 7F8U, 7F8Y and 7F8X. Atomic coordinates and cryo-EM density maps for the structures of inactive CCKBR–gastrin-Gi and CCKBR–gastrin-Gq have been deposited in the RCSB Protein Data Bank (PDB) under accession codes 7F8V and 7F8W, and the Electron Microscopy Data Bank (EMDB) under accession codes EMD-31493 and EMD-31494.

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Acknowledgements

This work was partially supported by the National Key R&D Programs of China 2018YFA0507000 (H.E.X., S.Z., M.-W.W., B.W. and Q. Zhao); CAS Strategic Priority Research Program XDB37030100 (B.W. and Q.Z.); the National Science Foundation of China grants 31825010 (B.W.), 31800621 (S.H.), 21704064 (Q. Zhou), 81773792 and 81973373 (D.Y.), 31770796 (Y.J.), 31971178 (S.Z.), 81872915 and 82073904 (M.-W.W.) and 81525024 (Q.Zhao); National Science and Technology Major Project of China – Key New Drug Creation and Manufacturing Program 2018ZX09735–001 (M.-W.W.) and 2018ZX09711002–002–005 (D.Y.); and Science and Technology Commission of Shanghai Municipality grant (18XD1404800). The synchrotron radiation experiments were performed at the BL41XU of SPring-8 with approval of the Japan Synchrotron Radiation Research Institute (Proposal no. 2019A2543, 2019B2543, 2019A2541 and 2019B2541). We thank the beam line staff members K. Hasegawa, N. Mizuno, T. Kawamura and H. Murakami of the BL41XU for help with X-ray data collection. The cryo-EM data were collected at the Cryo-Electron Microscopy Research Center, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences. The authors thank the staff at the SIMM Cryo-Electron Microscopy Research Center for their technical support.

Author information

Author notes

  1. These authors contributed equally: Xuefeng Zhang, Chenglin He, Mu Wang, Qingtong Zhou, Dehua Yang, Ya Zhu.

Authors and Affiliations

  1. State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Xuefeng Zhang, Mu Wang, Dehua Yang, Ya Zhu, Hui Zhang, Xiaojing Chu, Zhenlin Yang, Yi Jiang, Qiuxiang Tan, Shuo Han, H. Eric Xu, Ming-Wei Wang, Beili Wu & Qiang Zhao

  2. University of Chinese Academy of Sciences, Beijing, China

    Xuefeng Zhang, Mu Wang, Dehua Yang, Hui Zhang, H. Eric Xu, Ming-Wei Wang, Beili Wu & Qiang Zhao

  3. School of Pharmacy, Fudan University, Shanghai, China

    Chenglin He, Wenbo Feng & Ming-Wei Wang

  4. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Mu Wang, Suwen Zhao, Ming-Wei Wang & Beili Wu

  5. iHuman Institute, ShanghaiTech University, Shanghai, China

    Qingtong Zhou & Suwen Zhao

  6. Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai, China

    Qingtong Zhou & Ming-Wei Wang

  7. The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Dehua Yang, Antao Dai, Jia Wang & Ming-Wei Wang

  8. Novo Nordisk A/S, Måløv, Denmark

    Ulrich Sensfuss & Steffen Reedtz-Runge

  9. School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Beili Wu & Qiang Zhao

  10. Zhongshan Institute for Drug Discovery, The Institutes of Drug Discovery and Development, Chinese Aacademy of Sciences, Guangdong, China

    Qiang Zhao

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  1. Xuefeng Zhang
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Contributions

X.Z. optimized the construct, purified the CCKAR protein, performed crystallization trials, solved the structure, performed signaling assays and helped with manuscript preparation. C.H. optimized the construct, purified the CCKBR protein, performed crystallization trials, solved the structure, performed signaling assays and helped with manuscript preparation. M.W. processed the cryo-EM data and solved the structures of CCKBR. D.Y., W.F., A.D. and J.W. designed and performed the receptor binding and functional assays. Q. Zhou performed molecular dynamics simulation and docking studies, and helped with manuscript preparation. Y.Z. helped with signaling assay design and manuscript preparation. H.Z. collected X-ray diffraction data. X.C. helped with protein expression. Z.Y. participated in manuscript preperation. Y.J. solved the cryo-EM structures of CCKAR–G protein complexes. U.S. designed and synthesized the ligands. Q.T. asissited in the cryo-EM data collection. S.H. helped with structure determination. S.R.-R. helped with ligand selection and data analysis. H.E.X. helped with analysis of cryo-EM structures of CCKAR–G protein complexes and manuscript preparation. S.Z. oversaw molecular dynamics simulation and docking studies, and edited the manuscript. M.-W.W. oversaw the binding and signaling assays, and edited the manuscript. B.W. and Q. Zhao initiated the project, planned and supervised the research, and wrote the manuscript with inputs from all co-authors.

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Correspondence to Suwen Zhao, Ming-Wei Wang, Beili Wu or Qiang Zhao.

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Peer review information Nature Chemical Biology thanks Zhenhua Shao, Irina Tikhonova 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 Structures of the ligands in the CCKAR and CCKBR complexes and molecular dynamic simulations.

a, Devazepide. b, Lintitript. c, NN9056. d, Gastrin-17. e, CCK-8. f, CCK-8ns.

Extended Data Fig. 2 CCKAR crystals and lattice packing.

a–c, Crystals of CCKAR–T4L in complex with devazepide (a), lintitript (b) and NN9056 (c). d, Lattice packing of CCKAR–T4L–devazepide crystals with CCKAR depicted in green, T4L in grey and devazepide shown as spheres in blue. The main contacts contained nonpolar interactions among CCKAR molecules mediated by helices I and V and interactions between ECL2 and ECL3 of CCKAR and T4L. The packing patterns of CCKAR–T4L–lintitript and CCKAR–T4L–NN9056 are the same as CCKAR–T4L–devazepide.

Extended Data Fig. 3 Cryo-EM data processing of the CCKBR–gastrin-17–Gi2 and CCKBR–gastrin-17–Gq complexes.

a, Representative cryo-EM image and 2D averages of the CCKBR–gastrin-17–Gi2 complex. The cryo-EM results are repeated for three independent experiments. b, Representative cryo-EM image and 2D averages of CCKBR–gastrin-17–Gq complex. The cryo-EM results are repeated for three independent experiments. c, Workflow of cryo-EM data processing with cryo-EM map colored according to local resolution (Å) for the CCKBR–gastrin-17–Gi2 complex. d, Workflow of cryo-EM data processing with cryo-EM map colored according to local resolution (Å) for the CCKBR–gastrin-17–Gq complex. e, Gold-standard FSC curves and cross-validation of model to cryo-EM density map of CCKBR–gastrin-17–Gi2. FSC curve for the final model versus the final map, FSCwork curve, FSCfree curve and FSC curve are shown in black, red, blue and orange, respectively. f, Gold-standard FSC curves and cross-validation of model to cryo-EM density map of CCKBR–gastrin-17–GqiN. The curves are colored as the same as CCKBR–gastrin-17–Gi2.

Extended Data Fig. 4 Gi2 and Gq binding comparison in the CCKBR–gastrin-17 complexes.

a, Superposition of CCKBR–gastrin-17–Gi2 and CCKBR–gastrin-17–Gq complex structures. The structures are aligned based on receptor region. The CCKBRs are shown in bright-orange and tv_yellow cartoons for Gi2 and Gq complexes, respectively. The Gi2 trimers are shown in smudge, lime green and green cartoons, and the Gq trimers are shown in warm-pink, deep-salmon and salmon cartoons, respectively. Side view of Gi2 (b) and Gq (c) binding to the receptor from the same angle. The structures are colored according to panel (a).

Extended Data Fig. 5 Cut-view of orthosteric sites of CCKAR and CCKBR.

a, CCKAR–lintitript. b, CCKAR–devazepide. c, CCKAR–NN9056. d, CCKAR–CCK-8. e, CCKBR–gastrin-17.

Extended Data Fig. 6 Comparison of the CCKAR structures.

Extracellular (top), side (middle) and intracellular (bottom) views of agonist NN9056-bound (pale-yellow), antagonist-bound (pale-green for devazepide and wheat for lintitript), and both agonist and G protein bound CCKAR structures. G proteins are omitted for clarity. Conformational changes are indicated with black arrows.

Supplementary information

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Zhang, X., He, C., Wang, M. et al. Structures of the human cholecystokinin receptors bound to agonists and antagonists. Nat Chem Biol 17, 1230–1237 (2021). https://doi.org/10.1038/s41589-021-00866-8

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