High-resolution crystal structure of the human CB1 cannabinoid receptor

Abstract

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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Figure 1: Global structure of CB1 bound to taranabant.
Figure 2: Membrane-proximal N-terminal region of CB1.
Figure 3: Binding of taranabant to the CB1 receptor.
Figure 4: Docking of rimonabant and THC to the CB1 receptor.

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Acknowledgements

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).

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Authors

Contributions

Z.S. developed the CB1 construct and purification; expressed, purified and crystallized the receptor; collected diffraction data; and solved and refined the structure. J.Y. assisted with crystallographic refinement. K.C. performed ligand binding assays on CB1 constructs. M.G. carried out computational docking calculations. L.C. assisted with construct design and purification. J.W. performed and supervised computational docking calculations and molecular dynamics simulations. D.M.R supervised the overall project, assisted with collection of diffraction data, and wrote the manuscript.

Corresponding author

Correspondence to Daniel M. Rosenbaum.

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The authors declare no competing financial interests.

Additional information

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

Extended Data Figure 1 Differential scanning fluorimetry on purified CB1–PGS.

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.

Extended Data Figure 2 Ligand-binding properties of CB1 constructs.

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.

Extended Data Figure 3 Purification and crystallization of CB1(T210A)–PGS.

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.

Extended Data Figure 4 Packing and electron density in the CB1(T210A)–PGS crystals.

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, 2FoFc 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 2FoFc electron density (contoured at 1.5σ) for only the ligand taranabant (magenta sticks).

Extended Data Figure 5 Residues lining the putative lipid access channel of CB1.

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/).

Extended Data Figure 7 Molecular dynamics simulation of the CB1 structure.

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 2FoFc 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 2FoFc 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 2FoFc electron density (contoured at 1.0σ) shown for the N-terminal region, TM1 and AM6538.

Extended Data Table 1 Data collection and refinement statistics

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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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