XFEL structures of the human MT₂ melatonin receptor reveal the basis of subtype selectivity

The human MT₁ and MT₂ melatonin receptors^1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns³. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer³, and MT₂ has also been implicated in type 2 diabetes^4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT₂ receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon⁶ at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H208^5.46A (superscripts represent the Ballesteros–Weinstein residue numbering nomenclature⁷) and N86^2.50D, obtained in complex with 2-PMT. Comparison of the structures of MT₂ with a published structure⁸ of MT₁ reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [³H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT₁ and MT₂, but in addition the MT₂ structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT₂. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.