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

class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties. Access through your institution Buy or

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ACTIVITY AT THE CANNABINOID 1 RECEPTOR Article Open access 08 January 2025 STRUCTURAL BASIS FOR ACTIVATION OF CB1 BY AN ENDOCANNABINOID ANALOG Article Open access 09 May 2023 CANNABIDIOL

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Scholar  Download references 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. AUTHOR INFORMATION Author notes * Kiran Vemuri and Spyros P. Nikas:

These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * iHuman Institute, ShanghaiTech University, Shanghai, 201210, China Tian Hua, Yiran Wu, Lu Qu, Mengchen Pu, Kang

Ding, Suwen Zhao, Raymond C. Stevens & Zhi-Jie Liu * National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China Tian Hua, Lu

Qu & Zhi-Jie Liu * University of Chinese Academy of Sciences, Beijing, 100049, China Tian Hua, Lu Qu & Kang Ding * Center for Drug Discovery, Department of Pharmaceutical Sciences,

Kiran Vemuri, Spyros P. Nikas, Anisha Korde, Shan Jiang & Alexandros Makriyannis * Department of Chemistry and Chemical Biology, Northeastern University, Boston, 02115, Massachusetts,

USA Kiran Vemuri, Spyros P. Nikas, Anisha Korde, Shan Jiang & Alexandros Makriyannis * Departments of Molecular Medicine and Neuroscience, The Scripps Research Institute, Jupiter, 33458,

Florida, USA Robert B. Laprairie, Jo-Hao Ho & Laura M. Bohn * Departments of Biological Sciences and Chemistry, Bridge Institute, University of Southern California, Los Angeles, 90089,

California, USA Gye Won Han & Raymond C. Stevens * School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China Kang Ding, Suwen Zhao, Raymond C. Stevens &

 Zhi-Jie Liu * Complex Systems Division, Beijing Computational Science Research Center, Beijing, 100193, China Xuanxuan Li & Haiguang Liu * GPCR Consortium, San Marcos, 92078,

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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. CORRESPONDING

AUTHORS Correspondence to Suwen Zhao, Laura M. Bohn, Alexandros Makriyannis or Zhi-Jie Liu. ETHICS DECLARATIONS COMPETING INTERESTS 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. ADDITIONAL INFORMATION REVIEWER INFORMATION _Nature_ thanks G. Kunos and the

other anonymous reviewer(s) for their contribution to the peer review of this work. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published

maps and institutional affiliations. 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 |_F_o| − |_F_c|

omit maps of AM11542 and AM841 contoured at 3.0_σ_ 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 _Η__I_ 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 _Η__0_ and _Η__1_ of helices VI in the inactive-state and active-state β2-adrenergic receptor were

selected as reference to form a plane α. The vector _Η__I_ of helix VI of other structure was projected to the plane α as a new vector _Η__I__′_. The bending angle of each helix VI was then

defined by the angle between _Η__I′_ and _Η__0_. 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). SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION This file contains Supplementary Figures 1-2. (PDF 1249 kb) POWERPOINT SLIDES

POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE 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 Download

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