XFEL structures of the human MT 2 melatonin receptor reveal the basis of subtype selectivity.

The human MT ₁ and MT ₂ melatonin receptors ^1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns ³ . Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer ³ , and MT ₂ has also been implicated in type 2 diabetes ^4,5 . Here we report X-ray free electron laser (XFEL) structures of the human MT ₂ receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon ⁶ at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H208 ^5.46 A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature ⁷ ) and N86 ^2.50 D, obtained in complex with 2-PMT. Comparison of the structures of MT ₂ with a published structure ⁸ of MT ₁ reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [ ³ H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT ₁ and MT ₂ , but in addition the MT ₂ 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 MT ₂ . 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.