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

Glycine receptors (GlyR) are regulated by small-molecule binding at several allosteric sites. Endocannabinoids like tetrahydrocannabinol (THC) and N-arachidonyl-ethanol- amide (AEA) potentiate GlyR but their binding site is unknown. Using millisecond coarse-grained MD simulations powered by Martini 3 we have characterized their bind- ing site(s) at the Zebrafish GlyR-α1 with atomic resolution. Based on hundreds of thousand ligand-binding events, we find that cannabinoids bind at both intrasubunit and intersubunit sites in the transmembrane domain. For THC, intrasubunit binding is in excellent agreement with recent cryo-EM structures, while intersubunit binding recapitulates in full previous mutagenesis experiments. Surprisingly, AEA is found 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 results highlight an unanticipated complexity underlying allosteric regulation at synaptic receptors and establish an original simulation protocol for the identification and characterization of allosteric binding sites. ### Competing Interest Statement The authors have declared no competing interest.