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Serotonin (5-hydroxytryptamine; 5-HT) receptors modulate a variety of physiological processes ranging from perception, cognition and emotion to vascular and smooth muscle contraction, platelet aggregation, gastrointestinal function and reproduction. Drugs that interact with 5-HT receptors effectively treat diseases as diverse as migraine headaches, depression and obesity. Here we present four structures of a prototypical serotonin receptor—the human 5-HT2B receptor—in complex with chemically and pharmacologically diverse drugs, including methysergide, methylergonovine, lisuride and LY266097. A detailed analysis of these structures complemented by comprehensive interrogation of signaling illuminated key structural determinants essential for activation. Additional structure-guided mutagenesis experiments revealed binding pocket residues that were essential for agonist-mediated biased signaling and β-arrestin2 translocation. Given the importance of 5-HT receptors for a large number of therapeutic indications, insights derived from these studies should accelerate the design of safer and more effective medications.

Melatonin ( N -acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms 1 by synchronization to environmental cues and is involved in diverse physiological processes 2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function 3 . Melatonin is formed in the pineal gland in a light-regulated manner 4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness 5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT 1 ) and type 1B (MT 2 ) 3 , 6 . Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden 7 . Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids 8 , 9 , and is one of the most popular supplements in the United States 10 . Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT 1 in complex with four agonists: the insomnia drug ramelteon 11 , two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine 12 , 13 . The structure of MT 2 is described in an accompanying paper 14 . Although the MT 1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT 1 , access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT 1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors. The MT 1 melatonin receptor differs markedly from 5-HT receptors and shows atypical ligand entry; its structure with various ligands sheds light on receptor specificity.

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. Structural and functional studies show that the MT 2 melatonin receptor, unlike the MT 1 receptor, contains an extracellular opening for ligand entry, shedding light on receptor subtype specificity.

Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function . Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness by activating two high-affinity G-protein-coupled receptors, type 1A (MT ) and type 1B (MT ) . Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden . Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids , and is one of the most popular supplements in the United States . Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT in complex with four agonists: the insomnia drug ramelteon , two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine . The structure of MT is described in an accompanying paper . Although the MT and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT , access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

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