A millisecond coarse-grained simulation approach to decipher allosteric cannabinoid binding at the glycine receptor α1

Abstract

Glycine receptors (GlyR) are regulated by small-molecule binding at several allosteric sites. Cannabinoids like tetrahydrocannabinol (THC) and N-arachidonyl-ethanol-amide (AEA) potentiate the GlyR response but their mechanism of action is not fully established. By combining millisecond coarse-grained (CG) MD simulations powered by Martini 3 with backmapping to all-atom representations, we have characterized the cannabinoid-binding site(s) at the zebrafish GlyR-α1 active state with atomic resolution. Based on hundreds of thousand ligand-binding events, we find that cannabinoids bind to the transmembrane domain of the receptor at both intrasubunit and intersubunit sites. For THC, the intrasubunit binding mode predicted in simulation is in excellent agreement with recent cryo-EM structures, while intersubunit binding recapitulates in full previous mutagenesis experiments. Intriguingly, AEA is predicted to bind at the same intersubunit site despite the strikingly different chemistry. Statistical analyses of the ligand-receptor interactions highlight potentially relevant residues for GlyR potentiation, offering experimentally testable predictions. The predictions for AEA have been validated by electrophysiology recordings of rationally designed mutants. The results highlight the existence of multiple cannabinoid-binding sites for the allosteric regulation of GlyR and put forward an effective strategy for the identification and structural characterization of allosteric binding sites.

Fig. 1. Equilibrium distribution of cannabinoid ligands around GlyR.

A 2D-density maps (in nm⁻³) for THC (blue) and AEA (orange) in the lipid membrane split in upper and lower leaflets as shown in Supplementary Fig. 1. Note that the lower-density zones at the corners of the simulation box result from the re-centering the MD trajectory on the protein rather than a sampling issue. B 3D-density maps. The protein subunits are color-coded in green and white alternately to highlight the interfaces and distinguish between intrasubunit and intersubunit binding. The membrane environment is shown as a semi-transparent region. In all cases, darker colors correspond to binding hot spots.

Fig. 2. THC allosteric binding at GlyR.

A Specific binding modes are represented with associated probabilities. The dominant binding modes are color-coded. B Residence time (τ_r) distributions of the three most important binding events. The characteristic residence time per binding mode is given. The fit was done as described in Methods. C Atomistic representation of the dominant binding modes. Protein chains are color-coded in green and white to highlight adjacent subunits. The THC molecule is shown in sticks and color-coded following the scheme in panel (A). For each binding mode, the protein residues involved in the receptor-ligand recognition are highlighted in the insets.

Fig. 3. AEA allosteric binding at GlyR.

A Specific binding modes are represented with associated probabilities. The dominant binding modes are color-coded. B Residence time (τ_r) distributions of the four most important binding events. The characteristic residence time per binding mode is given. The fit was done as described in Methods. C Atomistic representation of GlyR-AEA interaction in the dominant binding modes. Protein chains are color-coded in green and white to highlight adjacent subunits, while the AEA molecule is shown in sticks and color-coded following the scheme in panel (A). For each binding mode, the protein residues involved in the receptor-ligand recognition are highlighted in the insets.

Fig. 4. GlyR-AEA contact analysis.

The analysis was carried out for the three dominant GlyR-AEA-binding modes predicted by CG/MD; see Fig. 3, green (mode 1), red (mode 2), and orange (mode 4). On the left-hand side, the average number of contacts per residue is plotted along the sequence of the protein. Gray boxes indicate protein stretches corresponding to the transmembrane helices M1–M4. On the right-hand side, the residues identified by the contact analysis are indicated on the protein structure. The color code is the same as that in Fig. 3.

Fig. 5. Mutational analysis to characterize the AEA-binding site(s) at human GlyR-α1.

A AEA potentiation (as peak of AEA-glycine current/peak glycine current) is shown for WT (n = 16), hS267A (n = 12), hI225A (n = 11), hS296A (n = 9), hW239F (n = 9), hS241A (n = 5), hI229A (n = 14), hK385A (n = 9), hF242A (n = 13), hW243F (n = 11), hF295A (n = 15), hL298A (n = 18), hL299A (n = 13). Data were shown as mean ± s.d. for (n) independent experiments. Electrophysiology experiments were performed on independent oocytes, from multiple different surgeries. Two-sided Mann–Whitney test ^**P = 0.0039 (hI225A), ^*P = 0.0196 (hS296A), ^****P ≤ 0.0001 (hS241A), ^*P = 0.0401(hF242A), ^****P ≤ 0.0001 (hW243F), ^****P ≤ 0.0001 (hF295A), ^***P = 0.0003 (hL298A), ^****P ≤ 0.0001 (hL299A). B The structural location of residues explored by mutational studies is highlighted on the cryo-EM structure of zebrafish GlyR-α1 solved in complex with THC. Amino acids represented as thick sticks correspond to residues whose substitution yields a significant effect on the potentiation by AEA in electrophysiology. The red and orange colors correspond, respectively, to amino acids at the lower and upper intersubunit binding sites, as identified by the CG/MD simulations. THC molecules and hS296 lining the intrasubunit allosteric site implicated in THC potentiation are shown in white.

Fig. 6. Comparison of cannabinoid-binding modes predicted by CG/MD with experiments.

A Superimposition of the cryo-EM structure of zebrafish GlyR-α1 solved in complex with THC (PDB ID: 7M6O) with the intrasubunit binding mode for THC predicted in simulation (Fig. 2, cyan). The heavy-atom RMSD between experimental and predicted ligand-binding modes is 2.7 Å. B Superimposition of X-ray structure of the GLIC-GABA_AR chimera solved in complex with tetrahydrodeoxycorticosterone (PDB ID: 5OSB) with the THC intersubunit binding mode predicted in simulation (Fig. 2, orange). C Superimposition of the X-ray structure of human GlyR-α₃ solved in complex with ivermectin (PDB ID: 5VDH) with the AEA upper intersubunit binding mode predicted in simulation (Fig. 3, orange).

Fig. 7. CG/MD simulation setup.

A The GlyR active state (PDB ID: 6PM6) was embedded in a POPC lipid membrane with 5% of cannabinoids (all-atom on the left, coarse-grained on the right). The protein is shown as white and green cartoons. The POPC polar heads are shown as blue and orange spheres, with the alkyl tails represented as a cyan surface. The cannabinoid ligands are shown in red. Ions are shown as green (Na⁺) and violet (Cl⁻) spheres, while water molecules are represented by a light-blue continuum. B On the left, the chemical structure of tetrahydrocannabinol (THC) and N-arachidonyl-ethanol-amide (AEA) are shown. All-atom (AA) representations of the two cannabinoids are given in the middle. Corresponding coarse-grained (CG) representations are given on the right, with beads represented as semi-transparent colored spheres.