Abstract
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.
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Acknowledgements
This work was supported by the NSF of China grant 31330019 (Z.-J.L.), the MOST of China grants 2014CB910400 (Z.-J.L.) and 2015CB910104 (Z.-J.L.), NSF of Shanghai 16ZR1448500 grant (S.Z.), Key R&D Program of China grant 2016YCF0905902 (S.Z.), NIH grants R01DA041435 (R.C.S., A.M.), P01DA009158 (A.M., L.M.B.), R37DA023142 (A.M.), NSF grants, Shanghai Municipal Government, ShanghaiTech University and GPCR Consortium. The diffraction data were collected at GM/CA@APS of Argonne National Laboratory, X06SA@SLS of the Paul Scherrer Insitute, and BL41XU@Spring-8 with JASRI proposals 2015B1031 and 2016A2731. We thank M. Wang, C.-Y. Huang, V. Olieric, M. Audet and M.-Y. Lee for their help with data collection, A. Walker for critical review of the manuscript, and F. Sun for high-resolution mass spectrometry analysis.
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Contributions
T.H.: crystallization, data collection, structure determination and analysis; K.V., S.P.N., S.J.: design, synthesis and characterization of ligands; Y.W.: docking, molecular dynamics simulation; L.Q., M.P.: data collection and processing, structure refinement; G.W.H., M.A.H.: structure refinement and data analysis. R.B.L. and J.-H.H.: functional studies, mutations; A.K.: radioligand binding assays; K.D.: structure analysis; X.L. and H.L.: molecular dynamics simulations; S.Z.: supervision of structure and simulation analysis; L.M.B.: design and supervision of functional and kinetic studies; A.M.: supervision on agonist conceptual design, synthesis and characterization; R.C.S.: project conception, data analysis supervision; Z.J.L.: design and supervision of experiments, data analysis; Z.J.L., T.H., R.C.S., A.M., L.M.B. and S.Z. wrote the manuscript with discussions and improvements from M.A.H., K.V., S.P.N. and Y.W.
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A.M. is a founder of MAKScientific, LLC. R.C.S. is a board member and shareholder with Birdrock Bio. The remaining authors declare no competing financial interests.
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Extended data figures and tables
Extended Data Figure 1 Synthesis of AM841 and AM11542.
Reagents and conditions: (a) CH3I, NaH, DMF, 0 °C to room temperature, 2 h, 95%; (b) DIBAL-H, CH2Cl2, −78 °C, 0.5 h, 87%; (c) Br− P+Ph3(CH2)5OPh, (Me3Si)2NK, THF, 0–10 °C, 30 min, then addition to 3, 0 °C to room temperature, 2 h, 96%; (d) H2, 10% Pd/C, AcOEt, room temperature, 2.5 h, 89%; (e) BBr3, CH2Cl2, −78 °C to room temperature, 6 h, 85%; (f) diacetates, p-TSA, CHCl3, 0 °C to room temperature, 4 days, 64%; (g) TMSOTf, CH2Cl2/MeNO2, 0 °C to room temperature, 3 h, 71%; (h) TBDMSCl, imidazole, DMAP, DMF, room temperature, 12 h, 85%; (i) Cl− Ph3P+CH2OMe, (Me3Si)2NK, THF, 0 °C to room temperature, 1 h, then addition to 9, 0 °C to room temperature, 1.5 h, 73%; (j) Cl3CCOOH, CH2Cl2, room temperature, 50 min, 95%; (k) K2CO3, EtOH, room temperature, 3 h, 84%; (l) NaBH4, EtOH, 0 °C, 30 min, 98%; (m) TBAF, THF, −40 °C, 30 min, 96%; (n) TMG-N3, CHCl3/MeNO2, room temperature, 18 h, 84%; (o) PPh3, CS2, THF, room temperature, 10 h, 76%; (p) (+)-cis/trans-p-mentha-2,8-dien-1-ol, p-TSA, benzene, reflux 4 h, 65%.
Extended Data Figure 2 Analytical size exclusion chromatography profile and crystals of CB1–AM11542/AM841 complex.
a, Analytical size exclusion chromatography and crystal image of the CB1–AM11542 complex. Scale bar, 70 μm. b, Analytical size exclusion chromatography and crystal image of the CB1–AM841 complex. Scale bar, 70 μm. c, The overall structures of CB1–AM11542 and CB1–AM841 complexes and crystal packing of CB1–AM11542; receptor is in orange (AM11542)/green (AM841) colour and the flavodoxin fusion protein is in purple-blue colour. The agonists AM11542 (yellow) and AM841 (pink) are shown in sticks representation. The four single mutations T2103.46A, E2735.37K, T2835.47V and R3406.32E are shown as green spheres in the CB1–AM11542 structure.
Extended Data Figure 3 Representative electron density of the CB1 agonists-bound structures and cholesterol binding sites.
a, The |Fo| − |Fc| omit maps of AM11542 and AM841 contoured at 3.0σ at 2.80 Å and 2.95 Å, respectively. b, The cholesterol binding site in the CB1–AM11542 structure (orange) with CB1–AM6538 structure (blue) superposed.
Extended Data Figure 4 Mutations of the CB1 receptor and the effects on agonist-induced activity as assessed by the forskolin-stimulated accumulation of cAMP.
a, Primers used to generate mutations in 3×HA–CB1 and validation of cell-surface expression of wild-type and mutant CB1 in CHO-K1 cell lines quantitative flow cytometry. b, Dose response studies of agonist (AM11542, AM841 and CP55,940) activity for each mutant compared to wild type (in blue filled circles) from Fig. 3c. c, Assessment of the effect of the individual point mutations that were made to stabilize the receptor, in absence of the flavodoxin insert, on receptor activity. All experiments were repeated at least three times, and error bars denote s.e.m. of duplicate measurements (parameters are in Extended Data Table 2).
Extended Data Figure 5 Docking poses of different cannabinoid receptor agonists and MD validation.
a–f, The r.m.s.d. values of ligand heavy atoms show that the docked poses are stable during the 1 μs molecular dynamics simulations: Δ9-THC (a), AEA (b), JWH-018 (c), HU-210 (d), 2-AG (e), WIN 55,212-2 (f). g, h, j, k, The poses of HU-210 (g), JWH-018 (h), 2-AG (j) and WIN 55,212-2 (k) are shown. i, The superimposition of HU-210 (yellow sticks) and HU-211 (blue sticks) in the binding pocket. The binding pose of HU-210 explains why HU-211, the enantiomer of HU-210, failed to stimulate CB1 because superimposed HU-211 on HU-210 shows severe clashes with H1782.65 in CB1.
Extended Data Figure 6 Structural conformation changes of solved agonist- and antagonist-bound class A GPCRs.
a, The pattern of r.m.s.d. values of transmembrane helices between agonist- and antagonist-bound class A GPCR structures. The structures used for analysis are the same as described in Extended Data Table 3. b, Measurement of the degree of helix VI bending observed in class A GPCRs structures. All structures were superimposed onto inactive-state β2-adrenergic receptor by UCSF Chimera. The direction of helices VI were defined by vectors ηi which starts from the centre of Cα of residues 6.45–6.48 to the centre of Cα of residues 6.29-30–6.32-33. The two vectors η0 and η1 of helices VI in the inactive-state and active-state β2-adrenergic receptor were selected as reference to form a plane α. The vector ηi of helix VI of other structure was projected to the plane α as a new vector ηi′. The bending angle of each helix VI was then defined by the angle between ηi′ and η0. The structures are: ETB (PDB code 5GLH), β1-adrenergic receptor (PDB code 2Y02), P2Y12 (PDB code 4PXZ), β2-adrenergic receptor (PDB code 3PDS), FFA1 (PDB code 4PHU), 5HT2B (PDB code 4IB4), 5HT1B (PDB code 4IAR), Rho (PDB code 2HPY), A2A (PDB code 3QAK), NTS1 (PDB code 4BUO), CB1 (bound to AM11542; PDB code 5XRA), μ-opioid receptor + nanobody (NB) (PDB code 5C1M), Rho + NB (PDB code 2X72), Rho + arrestin (PDB code 4ZWJ), M2R + NB (PDB code 4MQS), β2-adrenergic receptor + NB (PDB code 4LDL), A2A + mini-Gs (PDB code 5G53), β2-adrenergic receptor + Gs (PDB code 3SN6).
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Hua, T., Vemuri, K., Nikas, S. et al. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature 547, 468–471 (2017). https://doi.org/10.1038/nature23272
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DOI: https://doi.org/10.1038/nature23272
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