Abstract

The synthetic positive allosteric modulator (PAM) of cannabinoid receptor CB2, Ec21a, exhibits better subtype selectivity and receptor specificity over orthosteric ligands, which holds promise for treating neuropathic pain and seizure without causing psychotropic side effects mediated by CB1. The poor understanding of allosteric binding site and regulatory mechanism of Ec21a hinders further development of CB2 allosteric modulators. Here, we resolve the cryo-EM structure of CB2 in complex with PAM Ec21a and agonist CP55940, revealing a sandwich-like pattern of ECL2-Ec21a-CP55940, in which Ec21a binds above CP55940 acting as a “plug” and sterically hinders the dissociation of the CP55940. Through structural analysis and cell functional experiments, we find that Ec21a significantly enhances the activation efficiency of CP55940 via increasing and prolonging the interaction between CP55940 and CB2. Furthermore, by assessing the allosteric effects of Ec21a in combination with various agonists, we expand the potential range of ligand pairings and provide a structural framework for the design of bitopic ligands. Our findings address a gap in the understanding of the CB2 allosteric site and offer valuable guidance for the rational design of CB2 allosteric and bitopic ligands.