Neuromedin peptides, a structurally and functionally diverse neuropeptide family, consist of four groups of related peptides, the bombesin-like peptides (NmB and NmC), the kassinin-like peptides (NmK and NmL), a neurotensin-like peptide (NmN) and the neuromedin U group (NmU and NmS). Unlike other neuromedins, NmU peptides from different species share an identical C-terminal pentapeptide with a conserved C-terminal amidation. There are two subtypes of structurally related NmU peptides, NmU-25 and NmS-33, in humans. NmU-25 is ubiquitously distributed in the gastrointestinal tract, spinal cord, and central nervous system, while NmS-33 shows a more restricted distribution, predominantly expressed in the central nervous system. The system of NmU-25/NmS-33 implicates in the regulation of smooth muscle contraction, energy balance, feeding behavior, pronociceptive, and tumorigenesis. By activating two GPCRs, NMU1 and NMU2, NmU peptides are related to multiple pathophysiological roles in diabetes, metabolic disorder, inflammation, and cancer. NMU1 and NMU2 share almost 40% sequence identity and recognize NmU-25 and NmS-33 with high affinity. NMU1 is mainly found in the periphery tissues with the function of regulating intestinal motility and smooth muscle contraction, whereas NMU2 is predominantly expressed in the central nervous system and elicits a response to food intake and nociception.
To discover the biological functions of NmU peptides and develop practical drug candidates, several NmU peptide analogs with metabolic stability and NMU-subtype selectivity have been developed. One selective small molecular antagonist, R-PSOP, was identified with high potency and selectivity for NMU2 (K i = 52 nM) and served as a tool for further exploring the biological roles of NMU2. It has been shown that activation of NMU2 dramatically decreases body weight and food intake in mice while, in contrast, inhibition of NMU2 promotes weight gain and aspiration for obesogenic food. Unfortunately, drug development targeting NMU2 has been limited, partially due to the lack of structural information. To reveal the molecular details of ligand recognition and subtype-selectivity of NMUs, we report the cryo-EM structure of the NMU2–G i1 complex bound to the endogenous peptide NmU-25. Together with mutagenesis and molecular docking studies, the structure represents the key signature shared by NmU peptides which is a prerequisite for ligand recognition as well as the mechanism of ligand selectivity. Moreover, we also capture a specific G protein-coupling conformation of NMU2, providing a frame image for the variable activation process between GPCRs and G protein.
To facilitate expression and purification of the NmU-25–NMU2–G i1 complex, a hemagglutinin (HA) signal peptide followed by a flag tag was introduced at the N terminus of NMU2, while the flexible C-terminal residues of the receptor (Q356-T415) were replaced by a PreScission protease site followed by a twin-strep affinity tag. The addition of tags and deletion of flexible terminus have little effect on NmU-25-induced receptor signaling as indicated by bioluminescence resonance energy transfer Gα i1 βγ biosensor (TRUPATH) where the engineered construct showed a similar pEC 50 value compared to the wild-type (WT) receptor. Dominant-negative Gα i1 (DNGα i1) containing five mutations (S47C, G202T, G203A, E245A, and A326S) was used to improve the stability of the complex as these mutations lead to a preference for a nucleotide-free state, and prevent the dissociation of Gβγ from the heterotrimer. Over 8700 movies were collected, and the structure of the NmU-25–NMU2–G i1 complex was determined by cryo-EM single-particle analysis at a global nominal resolution of 3.3 Å. Each component can be modeled unambiguously with a clear and strong density map.
The overall structure of NMU2 possesses a canonical seven transmembrane helical domain similar to other solved peptide receptors of class A GPCRs with helix VIII unmodelled due to its flexibility upon activation. Two short antiparallel β-strands are formed in extracellular loop 2 (ECL2) and stabilized through a conserved disulfide bond between C119 3.25 and C204 ECL2 (superscript numbers represent Ballesteros–Weinstein nomenclature). To accommodate the peptide ligand, the extracellular part of NMU2 is more widely opened compared with other solved peptide-bound receptors, which share a high sequence similarity with NMU2. Compared with the inactive structure of NTS 1, NMU2 adopts a fully active conformation on the intracellular side that is stabilized by G protein coupling with a remarkable outward movement of helix VI (~10 Å, measured by C α of R 6.30) as well as the inward movement of helix VII (~4.5 Å, measured by C α of L 7.55). Besides the movement of transmembrane helices, many structural features also indicate that NMU2 is in an active state. W281 6.48, which is termed as “toggle switch” and significant for GPCR activation, is in an active-like conformation and induces a rotamer switch of F277 6.44 as well as the rearrangement of the “P 5.50-I 3.40-F 6.44” motif and initiates the outward movement of helices V and VI. Additionally, the disruption of the helices II–III–VII network leads to the collapse of the interactions between helices III and VII, causing the rearrangement of the “NPxxY” motif. To allow insertion of the α5 helix of G protein, R144 3.50 is released to form a hydrogen network with Y236 5.58 and Y327 7.53 to create a cavity for G protein coupling. All these residues share highly conserved sequence identity, indicating a conserved activation mechanism between NMUs.
