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Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1

Nature Chemical Biology volume 18, pages 831–840 (2022)Cite this article

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Abstract

Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.

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Fig. 1: Overall structure of the CB1 in complex with agonist and allosteric modulator.
Fig. 2: Characterization of ZCZ011 interactions with CB1.
Fig. 3: TM2 rearrangement is critical for CB1 activation.
Fig. 4: ZCZ011 PAM binding affects the conformational rearrangement of TM2 and CB1 active state.
Fig. 5: Cryo-EM structure of the CP55940–ZCZ011-bound CB1–Gi complex.
Fig. 6: The NAM ORG27569 hinders TM2 conformational rearrangement and affects CB1 activation.

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Data availability

Structural data have been deposited in the Protein Data Bank (PDB) with coordinate accession numbers 7FEE (crystal structure of the CB1 bound with CP55940 and ZCZ011) and 7WV9 (cryo-EM structure of the CP55940–ZCZ011-bound CB1–Gi complex), and the Electron Microscopy Data Bank (EMDB), accession number EMD-32850, is provided for the cryo-EM structure. All other data generated or analyzed during this study are included in this published article (and its Supplementary information files) or are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31972916, 81930125, 82130104, 32100988), Science and Technology Department of Sichuan Province 2020YJ0208 (Z.S.), 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYYC20023, ZYGD18001, ZYXY21001). Science and Technology Department of Chengdu (2019-YF05-00294-SN to Z.S.). We thank staff of the BL18U beamline at National Center for Protein Sciences Shanghai (NCPSS) and BL32XU beamline of Spring-8. The diffraction data collection was performed at the BL32XU of Spring-8 with approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal number 2019B2705). We thank J. Giraldo for providing MD-generated receptor conformations at the beginning of the project. This research used resources from the Duyu High Performance Computing Center, Sichuan University, and Big Data Platform at West China Hospital of Sichuan University (WCH-BDP).

Author information

Author notes

  1. These authors contributed equally: Xin Yang, Xuehui Wang, Zheng Xu, Chao Wu.

Authors and Affiliations

  1. State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China

    Xin Yang, Xuehui Wang, Zheng Xu, Chao Wu, Yangli Zhou, Yifei Wang, Guifeng Lin, Kan Li, Ming Wu, Anjie Xia, Jingming Liu, Lin Cheng, Jun Zou, Wei Yan, Zhenhua Shao & Shengyong Yang

  2. Division of Nephrology and Kidney Research Institute, West China Hospital, Sichuan University, Chengdu, Sichuan, China

    Xuehui Wang, Zheng Xu, Chao Wu, Lin Cheng, Wei Yan & Zhenhua Shao

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  1. Xin Yang
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Contributions

Z.S. initiated the project for allostery of CB1. S.Y. and Z.S. conceived and supervised the project. S.Y. and X.Y. designed molecular dynamics simulations. X.Y. performed simulations with the assistance of Y.W. and J.Z. X.Y. analyzed simulations with the assistance of K.L., M.W. and A.X. X.W. performed gene expression and protein purification, crystallization and diffraction data collection with assistance of G.L., Y.Z. and J.L. X.W. determined and analyzed the crystal structures with the assistance of L.C. X.W. designed the expression constructs, purified the ZCZ011–CP55940–CB1–Gi complexes and prepared the final samples for cryo-EM experiments. Z.X. determined the cryo-EM structure with the assistance of X.W. C.W. and Z.S. designed the cellular assays and analyzed results. S.Y., Z.S. with the assistance of X.Y., W.Y. and Z.X. wrote and revised the manuscript.

Corresponding authors

Correspondence to Zhenhua Shao or Shengyong Yang.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Francesco Gervasio, Aashish Manglik 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 Purification and crystallization of CB1-CP55940-ZCZ011 ternary complex.

(a) Snake representation of CB1 crystallization construct used in this study. (b) First derivative analysis of DSF data for purified CB1 constructs with CP55940-ZCZ011. PGS is a fusion protein. 5 M means S2033.39K, T2103.46A, E2735.37K, T2835.47V and R3406.32E mutations in CB1 receptor. (c) Superdex 200 gel-filtration trace of CB1 receptor in complex with CP55940 and ZCZ011. SDS-PAGE is for crystallization protein sample validation. Samples are prepared and repeated independently over three times (n = 3). (d) Crystal images for CB1-CP55940-ZCZ011 complex. The left image shown in visible light and the right shown under polarized light. Crystals can be formed over three independent experiments (n = 3). (e) Lattice packing of structure of CB1-CP55940-ZCZ011 complex.

Source data

Extended Data Fig. 2 PAM binding site analysis.

(a) The 2Fo-Fc map of PAM ZCZ011 and surrounding residues within 4 Å are contoured at 1.5 σ level. (b) Measurement of expression levels of the CB1 WT and indicated mutants by ELISA experiments. The mutant receptor expression was normalized to comparable levels as wild-type receptor expression. Values are means ± SEM (n = 4) from four independent experiments performed in triplicates. ns, no significance, *p < 0.1, **p < 0.01, ***p < 0.001 (one−way analysis of variance [ANOVA] followed by the Dunnett’s test, compared with the response of WT, p = 0.66, p = 0.09, p = 0.39, p < 0.001, p = 0.91, p = 0.19, p = 0.12, p < 0.01, p = 0.99, p < 0.001, p < 0.001, p = 0.18, p = 0.05, p = 0.14, p = 0.13, p = 0.08, p > 0.99, p < 0.001, p < 0.01 from left to right). (c) Effects of the S1732.60A in CB1 on CP55940 induced cAMP inhibition. Data are presented as the means ± SEM (n = 3) of three independent experiments performed in triplicate. (d) The inhibitory effect of CP55940 on forskolin-induced cAMP accumulation assay in the presence of or the absence of 0.5 μM PAM ZCZ011. F1913.27L, S1993.35T and V2494.58Y mutations decreased PAM potency significantly. Bars represent differences in calculated the ratio of the potency of CP55940 (EC50 [half maximal effective concentration]) in the presence of ZCZ011 or not for each mutation relative to WT of CB1. ns means not significant, **p < 0.01, ***p < 0.001 (one-way analysis of variance [ANOVA] followed by the Dunnett’s test, compared with the response of WT, p < 0.01, p = 0.91, p > 0.99, p < 0.001, p < 0.001 from left to right). All data are presented as the means ± SEM; n = 3.

Source data

Extended Data Fig. 3 Comparison of allosteric sites of CB1 with that in other GPCRs (PAR2 and GPR40).

(a) Analysis of sequence conservation in PAM site of CB1. (b) Structural comparison of PAM bound CB1 with allosteric modulator AZ3451 bound PAR2 (5NDZ). (c) Structural comparison of PAM bound CB1 with TAK-875 bound GPR40 (4PHU).

Extended Data Fig. 4 Structural comparison of an active and inactive state of CB1.

Comparison of agonist bound CB1 (PDB: 6KPG) with antagonist bound CB1 structures (PDB: 5TGZ), including C6.47W6.48xP6.50, P5.50-I3.40-F6.44 (L2865.50-V2043.40-L3526.44), N7.49P7.50xxY7.53, D3.49R3.50Y3.51 motifs.

Extended Data Fig. 5 Conformational changes in CB1 activation.

(a) Comparison of the active state CB1 bound agonist AM841 (salmon, PDB: 6KPG) with the inactive state CB1 bound antagonist AM6538 (slate, PDB: 5TGZ) reveals that the intracellular end of the TM6 helix moves outward significantly. (b) The upward rearrangement of TM3 upon CB1 activation (salmon, PDB: 6KPG). (c) The extracellular end of TM2 has inward movement, and its intracellular end is found to have downward rotation along the helical axis when comparing with inactive state of CB1 (slate, PDB: 5TGZ). In particular, F1552.42 undergoes a major conformational transition during receptor activation. (d) Structural superimposition reveals that the extracellular ends of TM1 and TM2 move into the core of helical bundles.

Extended Data Fig. 6 Structural comparison of in active and inactive state of GPCRs.

Structural comparison of TM2 conformation in active and inactive GPCRs. (a)The comparison reveals that there is less obvious conformational rearrangement of TM2 in most GPCRs, including aminergic receptors β2AR (active PDB: 6NI3, inactive PDB: 5D5A), M2 (active PDB: 6OIK, inactive PDB: 5ZKC), D2 (active PDB: 6VMS, inactive PDB: 7DFP), 5-HT2B (active PDB: 5TUD, intermedia: 6DRY) and H1 (active PDB: 7DFL, inactive PDB: 3RDZ); Nucleotide receptors A1R (active PDB: 6D9H, inactive PDB: 5N2S), Lipid receptors CB2 (active PDB: 6KPF, inactive PDB: 5ZTY) and EP4 (active PDB: 7D7M, inactive PDB: 5YWY); Chemokine receptor CXCR2 (active PDB: 6LFM, inactive PDB: 6LFL); Peptide receptors OPRD (active PDB: 6PT2, inactive PDB: 4RWD), AT1R (active PDB: 6OS0, inactive PDB: 4ZUD) and MC4 (active PDB: 7AUE, inactive PDB: 6W25). (b) The comparison reveals that there is more obvious conformational rearrangement of TM2 in most GPCRs, including OX2R (active PDB: 7L1V, inactive PDB: 6TPN), 5-HT2A (active PDB: 6WHA, inactive PDB: 6A94), A2A (active PDB: 6GDG, inactive PDB: 6ZDV), OPRM (active PDB: 6DDE, inactive PDB: 4DKL), OPRK (active PDB: 6B73, inactive PDB: 6VI4).

Extended Data Fig. 7 Traces during simulations of CB1-CP55940, starting from the crystal structure (PDB: 6KQI).

(a) RMSD of CP55940 from the initial crystal state during MD simulations of CB1 with bound CP55940. (b) RMSD of Cα atoms of CB1 TM6 from the initial crystal state. (c) RMSD of Cα atoms of TM helices from the initial crystal state. (d) Distance between ionic lock residues in CB1 (R2143.50 and D3386.30).

Source data

Extended Data Fig. 8 Representative snapshots during unbiased simulations of CB1-CP55940, starting from the crystal structure (PDB: 6KQI).

(a) Conformation comparison of representative snapshot in trajectory #2 (smudge) and CP99540 bound inactive CB1 (PDB: 6KQI) (gray). (b) Along the MD trajectories of CP55940-bound CB1, a small number of other different binding poses exist for CP55940.

Extended Data Fig. 9 ZCZ011 enhances CB1 binding to Gi protein.

(a) Antagonist AM6538 (blue) -bound CB1 (light blue) (PDB 5TGZ) orthotopic pockets expand and agonist CP55940 (magenta) bound CB1 (green-cyan) orthotopic pockets shrink. (b) ZCZ011 bound CB1 to enhance the interaction between S1522.39 and Gi D350.

Extended Data Fig. 10 CB1 receptor-Gi complex bound to CP55940 and ZCZ011 purification, cryo-EM data collection and cryo-EM map quality.

(a) Size-exclusion chromatography elution profiles of the purified receptor complex. (b) SDS-PAGE analysis of CB1 receptor-Gαo1-Gβγ-scFv16 complexes. Samples are prepared and repeated over three times. (c) Representative cryo-EM image micrographs of CB1 receptor-Gαo1-Gβγ-scFv16 complexes (left panel) and 2D class averages (right panel), from one of the total 5501 movies. (d) Flow chart of cryo-EM data analysis for the densities of ghrelin-ghrelin receptor-Go complex. The final resolution of the density is 3.3 Å. (e) The ‘gold-standard’ FSC curves, with the global resolution defined at the FSC = 0.143 is 3.3 Å for CP55940 and ZCZ011 bound CB1 receptor-Gi complex. (f) Representative cryo-EM density maps and fitted atomic models for all seven transmembrane helixes and the helix 8.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Figs. 1–19 and Notes 1 and 2.

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Unprocessed gel.

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Yang, X., Wang, X., Xu, Z. et al. Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1. Nat Chem Biol 18, 831–840 (2022). https://doi.org/10.1038/s41589-022-01038-y

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