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

ABSTRACT: 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.

SUBMITTER: Johansson LC

PROVIDER: S-EPMC6589158 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Johansson Linda C LC Stauch Benjamin B McCorvy John D JD Han Gye Won GW Patel Nilkanth N Huang Xi-Ping XP Batyuk Alexander A Gati Cornelius C Slocum Samuel T ST Li Chufeng C Grandner Jessica M JM Hao Shuming S Olsen Reid H J RHJ Tribo Alexandra R AR Zaare Sahba S Zhu Lan L Zatsepin Nadia A NA Weierstall Uwe U Yous Saïd S Stevens Raymond C RC Liu Wei W Roth Bryan L BL Katritch Vsevolod V Cherezov Vadim V

Nature 20190424 7755

The human MT1 and MT2 melatonin receptors1,2 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 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 ...[more]

PMID: 31019305

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Project description:Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)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 burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 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 MT1, 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 MT1 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.

Project description:Bombesin receptor subtype (BRS)-3, a G-protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin (Bn) receptor for gastrin-releasing peptide. There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control, motility, and tumor growth. BRS-3 has low affinity for all Bn-related peptides; however, recently synthetic high-affinity agonists, [d-Tyr(6)/d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), were described, but they are nonselective for BRS-3 over other Bn receptors. Based on these peptides, three BRS-3-selective ligands were developed: peptide 2, [d-Tyr(6)(R)-3-amino-propionic acid(11),Phe(13),Nle(14)]Bn(6-14); peptide 3, [d-Tyr(6),(R)-Apa(11),4Cl-Phe(13),Nle(14)]Bn(6-14); and peptide 4, acetyl-Phe-Trp-Ala-His-(tBzl)-piperidine-3 carboxylic acid-Gly-Arg-NH(2). Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this, we used a chimeric/site mutagenesis approach. Substitution of extracellular domain 2 (EC2) of BRS-3 by the comparable gastrin-releasing peptide receptor (GRPR) domain decreased 26-, 4-, and 0-fold affinity for peptides 4, 3, and 2. Substitution of EC3 decreased affinity 4-, 11-, and 0-fold affinity for peptides 2 to 4. Ten-point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107 (EC2-BRS-3) for lysine (H107K) (EC2-GRPR) decreased affinity (25- and 0-fold) for peptides 4 and 1; however, it could not be activated by either peptide. Its combination with Val101 (TM2), Gly112 (EC2), and Arg127 (TM3) resulted in complete loss-of-affinity of peptide 4. Receptor-modeling showed that each of these residues face inward and are within 4 A of the binding pocket. These results demonstrate that Val101, His107, Gly112, and Arg127 in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3 selectivity of peptide 4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/receptor interactions with peptide 4 are critical for both binding and activation. Furthermore, these result demonstrate that even though these three BRS-3-selective agonists were developed from the same template peptide, [d-Phe(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14), their molecular determinants of selectivity/high affinity varied considerably.

Dataset Information

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

Publications

XFEL structures of the human MT<sub>2</sub> melatonin receptor reveal the basis of subtype selectivity.

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