Jiang Yi/Xu Huaqiang from Shanghai Institute of Materia Medica, Nature Sub-Journal Reveal the Molecular Mechanism of Neuromodulin NMU/NMS Selectively Recognizing Neuromodulin U Receptor

iNatureNeuromedin U receptors (NMUR), including NMUR1 and NMUR2, are a group of Gq/11-coupled G protein-coupled receptors (GPCRs). NMUR1 and NMUR2 exert different pleiotropic physiological functions in peripheral tissues and the central nervous system (CNS), respectively, according to their different tissue distributions. These receptors are stimulated by two endogenous neuropeptides, neuromedin U and S (NMU and NMS), with similar binding affinities. NMURs have drawn attention as potential drug targets for obesity and inflammatory diseases. Specifically, selective agonists of NMUR2 in peripheral tissues showed promising long-term anti-obesity effects with fewer central nervous system-related side effects. However, the mechanisms of peptide binding specificity and receptor activation remain elusive. On April 19, 2022, the research team of Jiang Yi/Xu Huaqiang from Shanghai Institute of Materia Medica, Chinese Academy of Sciences published the research results entitled "Structural insights into the peptide selectivity and activation of human neuromedin U receptors" in Nature Communications. This work reports for the first time that two types of neuromedin U receptor 1/2 (NMUR1/2) bind endogenous polypeptide hormones, neuromedin U (NMU) and neuromedin S, respectively.
(Neuromedin S, NMS), and four near-atomic-resolution structures of the Gq protein signaling complex, revealing the molecular mechanisms of ligand-recognition selectivity and receptor activation unique to members of this family. NMU and NMS are important members of the human endogenous neuromedin family, and their carboxyl-terminal amino acid composition and terminal amidation modification (FLFRPRN-NH2) are highly conserved. Two types of neuromodulins exert diverse and complex physiological functions by activating NMUR1 and NMUR2 in human central and peripheral tissues. For example, NMU plays an important role in the immune regulation of the human gastrointestinal tract, and a large number of studies have confirmed that it is closely related to the occurrence of inflammation and the regulation of the activity of innate lymphocytes; NMS, as a neuropeptide highly expressed in the suprachiasmatic nucleus of the brain, has Mainly involved in regulating the expression of "clock" genes and maintaining normal biological rhythms; NMU and NMS, after activating NMURs, regulate energy balance through the regulation of the central nervous system and the regulation of peripheral gastrointestinal motility.
There are numerous preclinical studies demonstrating their superior therapeutic efficacy for obesity with fewer side effects. Therefore, NMURs have become one of the popular targets for the treatment of important diseases such as obesity, biological rhythm disorders and immune inflammation. Since the two types of NMURs show significantly different tissue distributions (NMUR1 is mainly distributed in peripheral tissues, while NMUR2 is mainly distributed in the central nervous system), targeting NMUR2 while regulating the body’s energy balance often has lower peripheral side effects such as diarrhea.
Therefore, selective targeting of NMUR2 subtypes has become an important strategy for the development of anti-obesity drugs. However, due to the current lack of high-precision structures of NMURs in complexes with endogenous polypeptide ligands in the activated state, ligand-specific recognition and receptor subtype selectivity, and molecular mechanisms of receptor activation remain unresolved, which is also a challenge for developing Selective drugs targeting NMURs pose great challenges. The research team used single-particle cryo-electron microscopy to analyze the four complex structures of NMUR1 and NMUR2 receptors that bind to NMU and NMS, respectively, and the downstream Gq protein, with a resolution of 2.8-3.2 angstroms (Fig.1a);

The high degree of conservation of the carboxyl-terminal P5P-R6P-N7P-NH2 sequence of polypeptide ligands and the mode of action of NMURs, and the decisive role of this sequence in receptor activation; three key amino acids in the conserved sequence of polypeptide ligands were confirmed from the structural point of view The interaction environment between (L2P-F3P-R4P) and the two NMURs subtypes is significantly different.
Combined with the relevant reports of molecular docking simulation and peptide design and modification, it is proposed that the difference in the interaction environment determines the ligand selectivity.

Theoretical, laid an important theoretical basis for the design of NMURs selective agonists (Fig.1b-d); elucidated the key role of the conserved salt bridge formed by the receptor's E3.33 and R6.5 in stabilizing the receptor, explaining the peptide Induced receptor R6.55 to wobble toward the receptor helix core triggers a novel mechanism of receptor activation by rearrangement of the downstream hydrophobic network and enriches our understanding of the diversity of mechanisms by which GPCRs couple Gq/11 proteins. Figure 1.
Cryo-EM structures of neuropeptide U receptors 1 and 2 in complex with neuropeptide U and neuropeptide S, respectively. Four cryo-EM structures of NMUR1/2 complexes with neuropeptide hormones NMU and NMS and their Gq proteins, respectively; bd.
Recognition of L2P-F3P-R4P sequences of NMU and NMS to ligand pocket residues of NMUR1 and NMUR2, respectively Pattern comparison. In 2020, the research team innovatively used the NanoBiT tethering method to stabilize the GPCR-G protein complex, and solved the structure of a class of neuropeptides, vasoactive intestinal peptide and its receptor VIP1R complex (Nat.Commun.2020). The NanoBiT tethering method has now been used to solve the structures of more than 40 GPCR complexes. The research team further included vasopressin V2 receptors (Cell Res.2021), ghrelin receptors (Nat.Commun.2021), bradykinin receptors B1R and B2R (Nat.Struct.Mol.Biol.2021 ), cholecystokinin receptor 1 (Nat.Chem.Biol.2021), galanin receptors GAL1R and GAL2R (Nat.Commun.2022), and thyrotropin-releasing hormone receptor (Cell Res.2022) and other important Neuropeptide GPCRs have carried out systematic structural and functional studies. This work further deepens our understanding of the mechanism of ligand regulation of neuropeptide GPCRs. The cryo-electron microscopy data in this study were collected on the cryo-electron microscopy platform of the Shanghai Institute of Materia Medica. You Chongzhao, a 2019 doctoral student of Shanghai Institute of Materia Medica, Zhang Yumu, a doctoral student jointly trained by ShanghaiTech University and Shanghai Institute of Materia Medica, and Xu Peiyu, research assistant of Shanghai Institute of Materia Medica, are the co-first authors of the paper. Researcher Jiang Yi and Xu Huaqiang are the co-corresponding authors, and Shanghai Institute of Materia Medica is the first completion unit. This work has been funded by the National Natural Science Foundation of China, the major projects of the Ministry of Science and Technology, and the major projects of Shanghai Municipal Science and Technology.