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

The human MT(1)(1) and MT(2)(2) melatonin receptors are G protein-coupled receptors (GPCRs) involved in the regulation of circadian rhythm and sleep patterns(3). Drug development efforts target both receptors for treatment of insomnia, circadian rhythm and mood disorders, and cancer(3), while MT(2) has also been implicated in type 2 diabetes (T2D)(4,5). Here we report the X-ray Free Electron Laser (XFEL) structures of the human MT(2) receptor in complex with 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 MT(2) structures with MT(1)(8) reveals that, despite the fact that the orthosteric ligand-binding site residues are conserved, there are notable conformational variations as well as differences in [(3)H]-melatonin dissociation kinetics that provide new insights into the selectivity between melatonin receptor subtypes. In addition to the membrane-buried lateral ligand entry channel that is also observed in MT(1), the MT(2) structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data supporting a prominent role for the intramembrane ligand entry in both receptors, while simultaneously suggesting the possibility of 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.