SUMMARY Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids, and is a promising therapeutic target for pain management, inflammation, obesity and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Highlightsand CB2-AM12033 are determined d Structural evidence of G protein selectivity by CB1 and CB2 is identified d MD simulations reveal the distinct binding behavior of HU308 in CB2 and CB1 d Cholesterol molecule as an endogenous allosteric modulator of CB1 is uncovered

Graphical AbstractHighlights d Crystal structure of human CB2 in complex with antagonist AM10257 is determined d A high degree of conformational similarity with the agonistbound CB1 is uncovered d The yin-yang relationship of CB2 and CB1 will facilitate the design of selective drugs In BriefThe structure of the human cannabinoid receptor CB2 reveals how small molecules affect CB2 differently than CB1 and point to principles that could inform rational and selective drug design. SUMMARYThe cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

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