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Ligand recognition and allosteric modulation of the human MRGPRX1 receptor

Nature Chemical Biology volume 19, pages 416–422 (2023)Cite this article

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Abstract

The human MAS-related G protein–coupled receptor X1 (MRGPRX1) is preferentially expressed in the small-diameter primary sensory neurons and involved in the mediation of nociception and pruritus. Central activation of MRGPRX1 by the endogenous opioid peptide fragment BAM8-22 and its positive allosteric modulator ML382 has been shown to effectively inhibit persistent pain, making MRGPRX1 a promising target for non-opioid pain treatment. However, the activation mechanism of MRGPRX1 is still largely unknown. Here we report three high-resolution cryogenic electron microscopy structures of MRGPRX1–Gαq in complex with BAM8-22 alone, with BAM8-22 and ML382 simultaneously as well as with a synthetic agonist compound-16. These structures reveal the agonist binding mode for MRGPRX1 and illuminate the structural requirements for positive allosteric modulation. Collectively, our findings provide a molecular understanding of the activation and allosteric modulation of the MRGPRX1 receptor, which could facilitate the structure-based design of non-opioid pain-relieving drugs.

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Fig. 1: Cryo-EM structures of MRGPRX1–Gq complexes.
Fig. 2: Interactions of the endogenous peptide agonist BAM8-22 with MRGPRX1.
Fig. 3: Structural basis for ML382 modulation.
Fig. 4: Interactions of compound-16 with MRGPRX1 receptor.
Fig. 5: Structural comparison of MRGPRX1 with MRGPRX2 and MRGPRX4.

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Data availability

The coordinate and cryo-EM map of MRGPRX1–Gq–BAM8-22, MRGPRX1–Gq–BAM8-22/ML382 and MRGPRX1–Gq–compound-16 have been deposited to the Protein Data Bank (Electron Microscopy Data Bank) database with accession codes 8DWC (EMD-27752), 8DWG (EMD-27753) and 8DWH (EMD-27754), respectively. The cryo-EM micrographs of MRGPRX1–Gq–BAM8-22, MRGPRX1–Gq–BAM8-22/ML382 and MRGPRX1–Gq–compound-16 have been deposited in the EMPIAR database (https://www.ebi.ac.uk/empiar/) with accession numbers EMPIAR-11183, EMPIAR-11188 and EMPIAR-11191, respectively. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Institutes of Health grants R01DA055656 and U24DK116195 to B.L.R. and B.K.S. as well as the Michael Hooker Distinguished Professorship to B.L.R. We thank J. Peck and J. Strauss of the University of North Carolina Cryo-EM Core Facility for their technical assistance with this project.

Author information

Author notes

  1. Jonathan F. Fay

    Present address: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA

  2. These authors contributed equally: Yongfeng Liu, Can Cao.

Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Yongfeng Liu, Can Cao, Xi-Ping Huang, Ryan H. Gumpper, Sheng-Luen Shih, Brian E. Krumm, Shicheng Zhang & Bryan L. Roth

  2. National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Yongfeng Liu, Xi-Ping Huang, Sheng-Luen Shih & Bryan L. Roth

  3. Department of Pharmaceutical Sciences, University of California, San Francisco School of Medicine, San Francisco, CA, USA

    Moira M. Rachman & Brian K. Shoichet

  4. Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Jonathan F. Fay

  5. Division of Chemical Biology and Medicinal Chemistry, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    Bryan L. Roth

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Contributions

Y.L. designed the experiments, generated the MRGPRX1 constructs for Sf9 insect cell expression, expressed and purified the MRGPRX1 signaling complexes for cryo-EM study, performed the BRET assays, analyzed the data, prepared the figures and tables and assisted in manuscript preparation. C.C. designed the experiments, assisted in the protein purification, built the models, refined the structures, analyzed the data and prepared the figures and the manuscript. J.F. prepared the cryo-EM grids, collected the cryo-EM data and performed the cryo-EM map calculation. R.H.G. and M.M.R. assisted in the validation of structures. X.-P.H. and Y.L. performed the GPCRome assays. S.-L.S. and Y.L. generated the MRGPRX1 mutations for functional assays. B.E.K designed the mini-Gαq protein construct and assisted in the functional data analysis. S.Z. assisted in the protein expression. B.K.S. provided valuable insights and edited the manuscript. B.L.R. supervised the overall project and edited the manuscript.

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Correspondence to Jonathan F. Fay or Bryan L. Roth.

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Extended data

Extended Data Fig. 1 Extended cryo-EM analysis of the MRGPRX1-Gq complex bound to BAM8-22, BAM8-22/ML382, and compound-16, respectively.

a, Representative cryo-EM micrograph (scale bar: 100 nm) of the MRGPRX1-Gq-BAM8-22, MRGPRX1-Gq-BAM8-22/ML382 and MRGPRX1-Gq-comppound-16 complexes. The exact number of movies and particles used for each complex are shown in Supplementary Fig. 2. The experiment was repeated three times with similar result. b, Histograms of defocus values for micrographs used in the single particle analysis (see Supplementary Table 4 for more details). c, Orientational distribution heat map. d, 2D plots of the gold standard Fourier shell correlation (GSFSC) between half maps (black) and FSC between model and the B-factor sharpened map for respective refined model (red) as calculated by phenix.mtirage. e, Local resolution heat-map calculated using the local windowed FSC method.

Extended Data Fig. 2 Electron microscopy density map of MRGPRX1-Gq-BAM8-22, MRGPRX1-Gq-BAM8-22/ML382, and MRGPRX1-Gq-compound-16 complexes.

a-c, EM density of the ligand, TM1-TM7 helices of MRGPRX1 and the α5 and αN helices of Gq of MRGPRX1-Gq-BAM8-22 (a), MRGPRX1-Gq-BAM8-22/ML382 (b) and MRGPRX1-Gq-compound-16 (c), respectively.

Extended Data Fig. 3 Comparison of MRGPRX1 PAM binding site with other GPCRs.

a-d, The binding modes of agonist and PAM of MRGPRX1 (a), M2R (PDB ID: 4MQT) (b), GPR40 (PDB ID: 5TZY) (c) and DRD1 (PDB ID: 7LJD) (d). The peptide agonist is shown as cartoon. Small molecule orthosteric agonists and PAMs are shown as sticks. Orthosteric agonist and PAM are colored by bule and yellow, respectively.

Extended Data Fig. 4 Mutations of MRGPRX1 allosteric site residues affect ML382 allosteric properties.

a-l, Representative Gq BRET2 dose–response curves of WT and mutations of key residues of MRGPRX1 in the presence of indicated concentration of the allosteric modulator ML382. Data are mean ± SEM of n = 3 biological replicates.

Source data

Extended Data Fig. 5 Structural comparison of the MRGPRX1-Gq-BAM8-22 complex with MRGPRX1-Gq-BAM8-22/ML382 complex.

a-c, The overall alignment of the MRGPRX1-Gq-BAM8-22 complex with MRGPRX1-Gq-BAM8-22/ML382 complex. Side-view to show the overall complexes (a). Extracellular view to show the overall similar peptide binding mode (b). Intracellular view to show the α5 binding pocket (c). d, Cross-section image to show the binding of BAM8-22 and ML382 in MRGPRX1. e, Cross-section image to show the binding of BAM8-22 alone in MRGPRX1.

Extended Data Fig. 6 Selectivity of compound-16 and ML382 among other GPCRs.

a-b, Screening of ML382 (a) and compound-16 (b) in single concentration (10 µM) at 318 receptors in PRESTO-Tango GPCRome assays. Dopamine (D2) with 100 nM quinpirole was used as the assay control (assay ctrl). The data are plotted as fold of basal activity and presented as mean ± SEM (n = 4). The ones with >3-fold of basal were took as pronounced hits. c-h, Follow-up dose response curves for the targets with > 3-fold increased activity. Known agonist for each receptor was used as positive controls, and all the results were normalized to these controls (c-g). For MRGPRX1, the results were plotted as fold of basal (g-h). All the data are presented as mean ± SEM of n = 3 independent experiments.

Source data

Extended Data Fig. 7 Structural comparison of MRGPRX1-Gq-compound-16 complex with MRGPRX1-Gq-BAM8-22/ML382 complex.

a, The orthosteric pocket for compound-16 (cyan) is larger than that of BAM8-22 (green). b-c, Structural superposition of MRGPRX1-Gq-compound-16 complex and MRGPRX1-Gq-BAM8-22/ML382 to show compound-16 has a weak contact with the allosteric modulator ML382. Side view (b). Top view (c). d, ML382 displays very weak PAM activity at compound-16 in BRET2 Gq activation assay. Data are mean ± SEM of n = 4 biological replicates.

Source data

Extended Data Fig. 8 Residue compositions of the extracellular and intracellular pockets of MRGPRX family receptors.

a, Structure of MRGPRX1 with the resides conserved in MRGPRX1, MRGPRX2 (PDB: 7S8N) and MRGPRX4 (PDB: 7S8P) colored in red. Figure is generated by ENDscript45. b-d, Residue compositions of the orthosteric pocket (b), allosteric pocket (c) and G protein interface (d) of the MRGPRX family receptors. The pocket residues are selected based on the MRGPRX1-Gq-BAM8-22/ML382 structure. The conserved residues are highlighted in green.

Extended Data Fig. 9 The G protein interface of MRGPRX1.

a, key interactions between the intracellular cavity of MRGPRX1 (green) and the α5 helix of Gq (red). The structure of MRGPRX1-Gq-BAM8-22/ML382 complex is used for structural analysis. b, Key interactions between the ICL2 of MRGPRX1 and the G protein. c-e, BRET2 validation of the intracellular cavity mutations of MRGPRX1 in presence of compound-16 (c), BAM8-22 alone (d) and BAM8-22 with 1 µM ML382 (e). f-h, BRET2 validation of the ICL2 mutations of MRGPRX1 in presence of compound-16 (f), BAM8-22 alone (g) and BAM8-22 with 1 µM ML382 (h). Data are mean ± SEM of n = 4 biological replicates.

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Liu, Y., Cao, C., Huang, XP. et al. Ligand recognition and allosteric modulation of the human MRGPRX1 receptor. Nat Chem Biol 19, 416–422 (2023). https://doi.org/10.1038/s41589-022-01173-6

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