Abstract
The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210.
Highlights
Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (؊)-⌬9-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-3566.48 and Trp-2795.43 is crucial for the Trp-3566.48 rotamer change toward receptor activation through the rigid-body movement of helix 6 (H6)
It appears that the hydrophobic pocket that interacts with the C3 alkyl chain of cannabinoids forms dynamically as the receptor shifts its equilibrium toward the active state
The results indicate the greatest decrease in HU210 binding affinity for this series was observed for the F1772.64A mutant receptor that bound HU210 with an affinity ϳ700-fold lower than the wild-type receptor (WT HU210 Ki ϭ 0.23 nM)
Summary
Generation of CB1 Receptor Mutants—Site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) was performed using the human CB1 cDNA cloned into pcDNA3.1. GTP␥S Binding Assay—GTP␥S binding assays were performed by incubating ϳ10 g of membrane in GTP␥S binding buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, and 100 mM NaCl) with at least nine concentrations of unlabeled HU210 (ranging from 10 pM to 10 M), 10 M GDP, 0.1% fatty acid-free BSA (w/v), and 0.1 nM [35S]GTP␥S (1,250 Ci/mmol; PerkinElmer Life Sciences) in a final volume of 200 l for 60 min at 30 °C. We checked the validity of the newly determined CHARMM parameters for describing HU210, CP55940, and ⌬9-THC by comparing molecular geometries from the MD simulations and key torsional energy barriers by CHARMM with those obtained by ab initio RHF/6 –31G* and MP2/6 – 31G* level calculations (supplemental Table 1 and supplemental Fig. 1).
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