Mechanism of agonist-induced activation of the human itch receptor MRGPRX1
Bing Gan, Leiye Yu, Haifeng Yang, Haizhan Jiao, Bin Pang,Yian Chen,Chen Wang, Rui Lv, Hongli Hu, Zhijian Cao, Ruobing Ren
Abstract
Mas-related G-protein-coupled receptors X1-X4 (MRGPRX1-X4) are 4 primate-specific receptors that are recently reported to be responsible for many biological processes, including itch sensation, pain transmission, and inflammatory reactions. MRGPRX1 is the first identified human MRGPR, and its expression is restricted to primary sensory neurons. Due to its dual roles in itch and pain signaling pathways, MRGPRX1 has been regarded as a promising target for itch remission and pain inhibition. Here, we reported a cryo-electron microscopy (cryo-EM) structure of Gq-coupled MRGPRX1 in complex with a synthetic agonist compound 16 in an active conformation at an overall resolution of 3.0 Å via a NanoBiT tethering strategy. Compound 16 is a new pain-relieving compound with high potency and selectivity to MRGPRX1 over other MRGPRXs and opioid receptor. MRGPRX1 was revealed to share common structural features of the Gq-mediated receptor activation mechanism of MRGPRX family members, but the variable residues in orthosteric pocket of MRGPRX1 exhibit the unique agonist recognition pattern, potentially facilitating to design MRGPRX1-specific modulators. Together with receptor activation and itch behavior evaluation assays, our study provides a structural snapshot to modify therapeutic molecules for itch relieving and analgesia targeting MRGPRX1.
Introduction
Itch is defined as the sensation that causes the desire to scratch the skin [1]. It is a common and frequently occurring symptom associated with many skin diseases among humans [2]. Numerous factors can induce itches, such as chemicals, insect bites, and even self-generated substances resulting from varied diseases [3]. Unfortunately, due to diverse inducements and complicated pathogenesis, treating itch in the clinic is still challenging, especially the chronic itch, which will devastate people and cause much suffering [4]. The itch can be generally divided into histaminergic and nonhistaminergic [5]. Usually, most histaminergic itch results in acute itch, whereas chronic itch is more probable to be nonhistaminergic [6]. Therefore, the well-developed antihistamine drugs are inefficient in chronic itch relieving, which suggests the significance of finding novel drug targets for chronic itch treatment [6].
Mas-related G-protein-coupled receptors (MRGPRs) have been recently identified as pruritogenic receptors mediating the nonhistaminergic itch [7]. The Mrgpr gene family encodes MRGPRs, a large family which comprises 27 and 8 members in mice and humans, respectively [7,8]. MRGPRX1-X4 are 4 primate-specific receptors, suggesting that the X subfamily may be a simplified alteration in human evolution [4]. MRGPRX1 is the first identified human MRGPR that expresses in dorsal root ganglia (DRG) and trigeminal ganglia (TG) specifically [4]. Compared with other MRGPRX members, MRGPRX1 stands out for its dual roles in mediating itch [9] and inhibiting persistent pain [10].
Material and methods
Construct design
The full-length wild-type (WT) human MRGPRX1 (residues M1-S382) was cloned into pFastBac vector with an HA signal peptide, an N-terminal Flag tag, and a C-terminal 10×His tag. A BRIL [22] protein was fused at the N-terminus of MRGPRX1 to improve the expression of the receptor. LgBit [23] was fused at the C-terminus of MRGPRX1 to stabilize the whole MRGPRX1-Gαq complex. There was no more modification for the MRGPRX1 sequence. Prof. H. Eric Xu from Shanghai Institute of Materia Medica donated the engineered Gαq chimera plasmid. This engineered Gαq [29,30] was designed based on a mini-Gαs/q71 [24,25] skeleton with the replacement of Gi1 N-terminus and the insertion of Gαi1 α-helical domain, thus providing possible binding sites for 2 antibody fragments scFv16 and Fab-G50 [26,27].
Results
The overall structure of Gq-coupled MRGPRX1 bound to compound 16
To improve receptor expression, we fused thermostabilized apocytochrome b562 (BRIL) [22] at the N-terminus of MRGPRX1. NanoBit tethering strategy [23] was used for the complex formation, with the LgBit and HiBit fused to the C-terminus of the receptor and Gβ subunit, respectively (S1A and S1B Fig). We used bioluminescence resonance energy transfer (BRET) assay to evaluate the impact of receptor modification on G protein coupling capability. The fusion of BRIL and LgBit to receptor only marginally affected receptor activity (S1C Fig and S1 Table). To further stabilize the complex, we used an engineered Gαq chimera in the complex assembly. The engineered Gαq chimera was designed based on the mini-Gαs/q71 [24,25] with several modifications (S1D Fig). Briefly, the N-terminal 1–18 residues of the mini-Gαs/q71 [24] were replaced by corresponding N-terminal sequences of the human Gαi1, while the α-helical domain of Gαi1 was subsequently inserted into the mini-Gαs/q71, thus providing possible binding sites for 2 antibody fragments scFv16 and Fab-G50 [26,27]. Additionally, 2 dominant-negative mutations (G203A and A326S) were introduced to decrease the affinity of nucleotide binding [28].
Discussion
In this study, we used the NanoBiT strategy to determine the structure of compound 16-bound MRGPRX1 in complex with Gαq via cryo-EM. We compared our compound 16-MRGPRX1-Gαq complex structure to the recently reported MRGPRX1-Gαq complex structure contributed by Liu and colleagues [16]. The overall structures are similar (S11A Fig), but the significant difference is the orientation of the phenylmethyl group in compound 16 (S11B and S11C Fig). Due to the better ligand density in our structure, compound 16 could be accommodated well with our proposed conformation. In contrast, the ligand leaves a part of the phenyl group out of the density map when we use Liu’s structure to fit the density. Our structure reveals the common feature of shallow, broad, and wide-open orthosteric pockets in all MRGPRX members. We speculated that this shallow and broad pocket might easily accommodate various small compounds with distinct scaffolds.
Acknowledgments
We are grateful to Prof. H. Eric Xu from the Shanghai Institute of Materia Medica (SIMM) for donating the engineered Gαq chimera plasmid and Prof. Sheng Wang from the Shanghai Institute of Biochemistry and Cell Biology for donating the plasmids for functional assays. We also appreciate Prof. Xinhua Lin from the School of Life Sciences Fudan University for providing the LB940 Mithras plate reader (Berthold Technologies) for BRET assay. We thank Kobilka cryo-EM Center at the Chinese University of Hong Kong, Shenzhen, for supporting EM data collection.
Citation: Gan B, Yu L, Yang H, Jiao H, Pang B, Chen Y, et al. (2023) Mechanism of agonist-induced activation of the human itch receptor MRGPRX1. PLoS Biol 21(6): e3001975. https://doi.org/10.1371/journal.pbio.3001975
Academic Editor: Raimund Dutzler, University of Zurich, SWITZERLAND
Received: December 6, 2022; Accepted: May 31, 2023; Published: June 22, 2023
Copyright: © 2023 Gan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files. Cryo-EM maps and atomic models have been deposited in the Electron Microscopy Data Bank under accession codes EMD-34833 and in the Protein Data Bank under accession code 8HJ5.
Funding: This work was supported by funds from National Natural Science Foundation of China project 32070525 (to Z.J.C.); Shenzhen Science and Technology Program project JCYJ20220530140800001 (to Z.J.C.). B. G. were supported by Ganghong Youth Scholarship at the Chinese University of Hong Kong, Shenzhen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BRET, bioluminescence resonance energy transfer; CQ, chloroquine; cryo-EM, cryo-electron microscopy; DRG, dorsal root ganglia; HBSS, Hank’s balanced salt solution; LMNG, lauryl maltose neopentylglycol; MRGPR, Mas-related G-protein-coupled receptor; TG, trigeminal ganglia; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1; WT, wild type; μOR, μ opioid receptor.
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001975#abstract0
