The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ9-tetrahydrocannabinol (THC)1. The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain2, epilepsy3, obesity4, and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.
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We thank the staff of the GM/CA-CAT beamline 23ID at the Advanced Photon Source (APS) for support during data collection. This project was supported by Welch Foundation grant (I-1770 to D.M.R) and a Packard Foundation Fellowship (D.M.R.). APS is a US Department of Energy Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory (DE-AC02-06CH11357).
The authors declare no competing financial interests.
Reviewer Information Nature thanks A. Christopoulos, G. Kunos and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Raw differential scanning fluorimetry traces of the receptor in the apo state or bound to each antagonist. b, First derivative analysis of data in a.
a, Saturation binding of the antagonist [3H]SR141716A (tritiated rimonabant radioligand) to wild-type CB1, CB1–PGS, and CB1(T210A)–PGS. Error bars represent s.d. for three separate experiments, each performed in duplicate. The fitted Kd values (± s.e.m.) for these three constructs are 4.8 ± 0.7 nM, 6.3 ± 0.6 nM, and 4.4 ± 0.5 nM, respectively. b, Competition binding of taranabant to the wild-type CB1 receptor, CB1–PGS, and CB1(T210A)–PGS. Error bars represent s.d. for three separate experiments, each performed in duplicate. The Ki values (± s.e.m.) of the three constructs for taranabant are 0.94 ± 0.17 nM, 1.10 ± 0.16 nM, and 0.91 ± 0.16 nM, respectively. c, Competition binding of the agonist CP55940 to the wild-type CB1 receptor, CB1–PGS, and CB1(T210A)–PGS. Error bars represent s.d. for three separate experiments, each performed in duplicate. The Ki values (± s.e.m.) of the three constructs for CP55940 are 53 ± 12 nM, 230 ± 43 nM, and 384 ± 62 nM, respectively.
a, Superdex 200 gel-filtration trace of receptor after Ni immobilized metal-affinity chromatography (IMAC) and M1 anti-Flag chromatography (see Methods). b, SDS–PAGE analysis of samples at different stages of purification. The five lanes from left to right are: markers (molecular mass in kDa at left); IMAC/Flag-purified receptor; same sample after treatment with PNGaseF; receptor after TEV protease cleavage (removing 89 N-terminal amino acids); final sample after Superdex 200 gel filtration. c, Light microscopy image showing examples of LCP microcrystals of CB1(T210A)–PGS used to collect diffraction data.
a, Lattice packing interactions in the monoclinic crystals of CB1(T210A)–PGS. Protomers are shown as ribbons, with the receptor component of the fusion protein coluored teal and the PGS domain coloured grey. b, 2Fo − Fc electron density map (contoured at 1.2σ) of taranabant and the surrounding ligand-binding residues. Protein and ligand are represented as yellow sticks. c, Stereo view of 2Fo − Fc electron density (contoured at 1.5σ) for only the ligand taranabant (magenta sticks).
The receptor is shown as a teal transparent surface, and taranabant is in magenta spheres. The three residues lining the channel are shown as orange sticks and their solvent-accessible surfaces are coloured orange.
Extended Data Figure 6 Sequence alignment of the membrane-proximal N-terminal region of CB1 from different vertebrate species.
‘Frog’ is Xenopus laevis. The red bar (top) indicates the part of this region that is structured and visible in the electron density of the CB1 crystals. The blue box denotes positions that make contact with taranabant. Alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
a, A 60-ns molecular dynamics simulation of the CB1 receptor (after removing the PGS fusion protein) with taranabant present. Black trace is for the entire receptor, red trace is for only the structured membrane-proximal N-terminal region. b, 60-ns molecular dynamics simulation of the CB1 receptor without a ligand present. Black trace is for the entire receptor, red trace is for only the structured membrane-proximal N-terminal region.
Extended Data Figure 8 Sequence alignment of the entire sequence of CB1 from several different species, along with human CB2.
The blue boxes denote positions that make contact with taranabant within a 4 Å cut-off. The alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
Extended Data Figure 9 Comparison of the structures of CB1 bound to taranabant and CB1 bound to AM6538 (ref. 34; PDB accession 5TGZ).
a, Superposition of the two CB1 structures viewed from the extracellular space. The taranabant-bound structure is shown as a teal cartoon (ligand as magenta sticks), while the AM6538-bound structure is shown as a gold cartoon (ligand as green sticks). b, Comparison of 2Fo − Fc electron density (contoured at 1.5σ) for the ligands in each structure. On the left is taranabant from the current structure, on the right is AM6538 from ref. 34. c, Comparison of the membrane-proximal N-terminal regions in each structure. On the left is a side view of CB1 from the current structure, with 2Fo − Fc electron density (contoured at 1.0σ) shown for the N-terminal region, TM1, and taranabant. On the right is the analogous side view of CB1 from ref. 34 (gold cartoon), with 2Fo − Fc electron density (contoured at 1.0σ) shown for the N-terminal region, TM1 and AM6538.
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Shao, Z., Yin, J., Chapman, K. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–606 (2016). https://doi.org/10.1038/nature20613
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