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.
ASJC Scopus subject areas