Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Crystal structures of agonist-bound human cannabinoid receptor CB1

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Synthesis and pharmacological characterization of AM11542 and AM841.
Figure 2: Overall structures of CB1–AM11542 and CB1–AM841 complexes.
Figure 3: AM11542 binding pocket analysis and molecular docking of Δ9-THC and AEA.
Figure 4: Structural comparison of agonist- and antagonist-bound CB1.

Accession codes

Primary accessions

Protein Data Bank

References

  1. Mechoulam, R., Hanuš, L. O., Pertwee, R. & Howlett, A. C. Early phytocannabinoid chemistry to endocannabinoids and beyond. Nat. Rev. Neurosci. 15, 757–764 (2014)

    CAS  Article  Google Scholar 

  2. Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762 e714 (2016)

    CAS  Article  Google Scholar 

  3. Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. 25 (ed. Sealfon Stuart, C. ) 366–428 (Academic, 1995)

  4. Lemberger, L. Potential therapeutic usefulness of marijuana. Annu. Rev. Pharmacol. Toxicol. 20, 151–172 (1980)

    CAS  Article  Google Scholar 

  5. Li, H.-L. An archaeological and historical account of cannabis in China. Econ. Bot. 28, 437–448 (1973)

    Article  Google Scholar 

  6. Makriyannis, A. 2012 Division of Medicinal Chemistry Award Address. Trekking the cannabinoid road: a personal perspective. J. Med. Chem. 57, 3891–3911 (2014)

    CAS  Article  Google Scholar 

  7. Shao, Z. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–606 (2016)

    CAS  ADS  Article  Google Scholar 

  8. Nikas, S. P. et al. The role of halogen substitution in classical cannabinoids: a CB1 pharmacophore model. AAPS J. 6, e30 (2004)

    Article  Google Scholar 

  9. Nikas, S. P. et al. Novel 1′,1′-chain substituted hexahydrocannabinols: 9β-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist. J. Med. Chem. 53, 6996–7010 (2010)

    CAS  Article  Google Scholar 

  10. Xie, X. Q., Melvin, L. S. & Makriyannis, A. The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. J. Biol. Chem. 271, 10640–10647 (1996)

    CAS  Article  Google Scholar 

  11. Makriyannis, A. & Rapaka, R. S. The medicinal chemistry of cannabinoids: an overview. NIDA Res. Monogr. 79, 204–210 (1987)

    CAS  PubMed  Google Scholar 

  12. Ahn, K. H., Bertalovitz, A. C., Mierke, D. F. & Kendall, D. A. Dual role of the second extracellular loop of the cannabinoid receptor 1: ligand binding and receptor localization. Mol. Pharmacol. 76, 833–842 (2009)

    CAS  Article  Google Scholar 

  13. Feigenbaum, J. J. et al. Nonpsychotropic cannabinoid acts as a functional N-methyl-d-aspartate receptor blocker. Proc. Natl Acad. Sci. USA 86, 9584–9587 (1989)

    CAS  ADS  Article  Google Scholar 

  14. Mechoulam, R. et al. Enantiomeric cannabinoids: stereospecificity of psychotropic activity. Experientia 44, 762–764 (1988)

    CAS  Article  Google Scholar 

  15. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012)

    CAS  ADS  Article  Google Scholar 

  16. Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011)

    CAS  ADS  Article  Google Scholar 

  17. Singh, R. et al. Activation of the cannabinoid CB1 receptor may involve a W6.48/F3.36 rotamer toggle switch. J. Pept. Res. 60, 357–370 (2002)

    CAS  Article  Google Scholar 

  18. Tiburu, E. K. et al. Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments. Biochem. Biophys. Res. Commun. 384, 243–248 (2009)

    CAS  Article  Google Scholar 

  19. Zhang, K. et al. Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature 509, 115–118 (2014)

    CAS  ADS  Article  Google Scholar 

  20. Zhang, J. et al. Agonist-bound structure of the human P2Y12 receptor. Nature 509, 119–122 (2014)

    CAS  ADS  Article  Google Scholar 

  21. Nikas, S. P. et al. A concise methodology for the synthesis of (−)-Δ9-tetrahydrocannabinol and (−)-Δ9-tetrahydrocannabivarin metabolites and their regiospecifically deuterated analogs. Tetrahedron 63, 8112–8113 (2007)

    CAS  Article  Google Scholar 

  22. Kulkarni, S. et al. Novel C-ring-hydroxy-substituted controlled deactivation cannabinergic analogues. J. Med. Chem. 59, 6903–6919 (2016)

    CAS  Article  Google Scholar 

  23. D’Antona, A. M., Ahn, K. H. & Kendall, D. A. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry 45, 5606–5617 (2006)

    Article  Google Scholar 

  24. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protocols 4, 706–731 (2009)

    CAS  Article  Google Scholar 

  25. Cherezov, V. et al. Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 microm size X-ray synchrotron beam. J. R. Soc. Interface 6 (suppl. 5), S587–S597 (2009)

    CAS  Article  Google Scholar 

  26. Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012)

    CAS  Article  Google Scholar 

  27. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  28. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  29. Adams, P. D . et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  30. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

    CAS  Article  Google Scholar 

  31. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  32. Friesner, R. A . et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 47, 1739–1749 (2004)

    CAS  Article  Google Scholar 

  33. Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49, 6177–6196 (2006)

    CAS  Article  Google Scholar 

  34. Halgren, T. A . et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 47, 1750–1759 (2004)

    CAS  Article  Google Scholar 

  35. Abraham, M. J. et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015)

    ADS  Article  Google Scholar 

  36. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

  37. Sousa da Silva, A. W. & Vranken, W. F. ACPYPE – AnteChamber PYthon Parser interfacE. BMC Res. Notes 5, 367 (2012)

    Article  Google Scholar 

  38. Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000)

    CAS  ADS  Article  Google Scholar 

  39. Skjærven, L., Yao, X. Q., Scarabelli, G. & Grant, B. J. Integrating protein structural dynamics and evolutionary analysis with Bio3D. BMC Bioinformatics 15, 399 (2014)

    Article  Google 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

Authors and Affiliations

Authors

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.

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 |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.

af, 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).

Extended Data Table 1 Data collection and structure refinement statistics
Extended Data Table 2 Mutations analysis of changes in pEC50 and Emax
Extended Data Table 3 Binding pocket volume comparison and r.m.s.d. analysis of solved representative agonist- and antagonist-bound pairs of seven class A GPCRs

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-2. (PDF 1249 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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 citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature23272

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing