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). 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 state, 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 Phe200^3.36 and Trp356^6.48 (superscripts denote Ballesteros-Weinstein numbering) 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.

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 T210^3.46A, E273^5.37K, T283^5.47V and R340^6.32E 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 3.0σ 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 H178^2.65 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 C_α of residues 6.45–6.48 to the centre of C^α 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 η_i of helix VI of other structure was projected to the plane α as a new vector ${\mathit{\eta }}_{\mathit{i}}^{\prime }$. The bending angle of each helix VI was then defined by the angle between ${\mathit{\eta }}_{\mathit{i}}^{\prime }$ and η₀. The structures are: ET^B (PDB code 5GLH), β₁-adrenergic receptor (PDB code 2Y02), P_2Y12 (PDB code 4PXZ), β₂-adrenergic receptor (PDB code 3PDS), FFA1 (PDB code 4PHU), 5HT_2B (PDB code 4IB4), 5HT_1B (PDB code 4IAR), Rho (PDB code 2HPY), A_2A (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), A_2A + mini-G_s (PDB code 5G53), β₂-adrenergic receptor + G_s (PDB code 3SN6).

Figure 1. Synthesis and pharmacological characterization of AM11542 and AM841

a, Synthesis of AM11542 and AM841 (Extended Data Fig. 1) with radioligand binding affinity against [³H]CP55,940. K_i, inhibition constant. b, Cell membranes were pretreated with CP55,940 (4 nM), AM11542 (1 nM), AM841 (10 nM) or buffer (control) for 1 h, washed, and then subjected to [³H]CP55,940 binding for 1 h. c, B_max values (maximal binding capacity) were calculated from b and in experiments where the incubation time of radioligand was increased following the washing. Pretreatment with either AM11542 or AM841 prevents radioligand binding after 1 h incubation (control: versus AM841: ##P < 0.01; versus AM11542 * * * P < 0.001), while AM11542 prevents radioligand binding at all later time points tested (control versus AM11542: * P < 0.05, * * P < 0.01). Displacement of [³H]CP55,940 binding in the presence of AM11542 (versus control: * * * P < 0.001) and AM841 (versus control: ###P < 0.001) are shown for comparison. (Student’s t-test versus control at each time point; data are mean ± s.e.m.; n = 3–6.) d, Agonist activity measured as the inhibition of forskolin-stimulated cAMP accumulation. Data are mean ± s.e.m. of n ≥ 6 independent experiments. %E_max, percentage of maximum response; EC₅₀, half-maximum effective concentration.

Figure 2. Overall structures of CB₁–AM11542 and CB₁–AM841 complexes

a, Superposition of CB₁–AM11542 and CB₁–AM841 structures with the surface outlined by an orange line. CB₁ is shown in orange and green cartoon with ligands AM11542 (yellow sticks) and AM841 (pink sticks). b, Comparison of agonist-bound (orange cartoon) and antagonist-bound (blue cartoon) CB₁ ligand-binding pockets. AM11542 (yellow) and AM6538 (green) are shown in sticks and sphere representations. c, d, The shape of AM11542 (c) and AM6538 (d) binding pockets are shown in surface representation.

Figure 3. AM11542 binding pocket analysis and molecular docking of Δ⁹-THC and AEA

a, Key residues (deepteal sticks) involved in AM11542 (yellow sticks) binding. b, Binding pose comparison of AM11542 (yellow), AM6538 (green) and ML056 (magenta) in their receptors, which are shown in orange, blue and grey cartoon, respectively. Phe170^2.57 and Phe174^2.61 in AM11542 and AM6538 complexes are shown in blue sphere and blue sticks, respectively. c, Certain mutations on CB₁ significantly decreased potency in the cAMP assay compared to agonist-response at the wild-type (WT) receptor as determined by comparison of negative logarithm of the EC₅₀ (pEC₅₀) values by two-way analysis of variance (ANOVA) without repeated measures followed by Dunnett’s post hoc test (comparing each drug to its effect in the wild type: * * * * P < 0.0001, data are mean ± s.e.m., n ≥ 3) (Extended Data Table 2). d, Summary of receptor interactions of AM11542. Purple ball (positive charged interaction); cyan ball (polar interaction); green ball (hydrophobic interaction); purple arrow (H-bond); green line (π–π stacking) e, f, The docking pose of Δ⁹-THC (e, blue sticks) and AEA (f, pink sticks).

Figure 4. Structural comparison of agonist-and antagonist-bound CB₁

a, Side view of the CB₁–AM11542 complex (receptor as orange and ligand as yellow) and CB₁–AM6538 complex (receptor in blue and ligand as green). b, The extracellular (left) and intracellular (right) views of the compared receptors. c, The ‘twin toggle switch’, Phe200^3.36/Trp356^6.48, is shown in sticks and spheres. Colour scheme as in a. d, Rearrangement of DRY (left) and NPXXY motifs (right) in agonist- and antagonist-bound CB₁ structures.