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Structure, function and pharmacology of human itch receptor complexes

Abstract

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals1,2. As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions3,4,5,6. MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2–Gi1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2–Gi1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp6.48 to Gly6.48 (superscript annotations as per Ballesteros–Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the Gi trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions.

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Fig. 1: Cryo-EM structure of MRGPRX2–Gi complexes and the C48/80-binding pocket.
Fig. 2: Consensus peptidic motif recognized by MRGPRX2.
Fig. 3: Activation mechanism of MRGPRX2.
Fig. 4: The coupling of MRGPRX2 to Gi.

Data availability

The cryo-EM density maps for the PAMP-12–MRGPRX2–Gi1 state 1 complex, PAMP-12–MRGPRX2–Gi1 state 2 complex, SP–MRGPRX2–Gi1 complex, C14circular–MRGPRX2–Gi1 complex, C14linear–MRGPRX2–Gi1 complex, C48/80–MRGPRX2–Gi1 state 1 complex and C48/80–MRGPRX2–Gi1 state 2 complex have been deposited at the EMDB (Electron Microscopy Data Bank) under accession codes EMD-32131, EMD-32132, EMD-31923, EMD-31922, EMD-32136, EMD-32138 and EMD-31918, respectively. The local maps of the receptor and ligand regions of the PAMP-12–MRGPRX2–Gil complex, C14linear–MRGPRX2–Gil complex and C48/80–MRGPRX2–Gil complex have been deposited at the EMDB (Electron Microscopy Data Bank) under accession codes EMD-32133, EMD-32137 and EMD-32139, respectively. The coordinates for the model of the PAMP-12–MRGPRX2–Gi1 state 1 complex (only receptor and Gil), PAMP-12–MRGPRX2–Gi1 state 2 complex (only receptor and Gil), PAMP-12–MRGPRX2complex (only ligand and receptor), SP–MRGPRX2–Gi1 complex, C14circular–MRGPRX2–Gi1 complex, C14linear–MRGPRX2–Gi1 complex (only receptor and Gil), C14linear–MRGPRX2 complex (only ligand and receptor), C48/80–MRGPRX2–Gi1 state 1 complex, C48/80–MRGPRX2–Gi1 state 2 complex and C48/80–MRGPRX2 complex (only ligand and receptor) have been deposited at the PDB under accession codes 7VUY, 7VUZ, 7VV0, 7VDM, 7VDL, 7VV3, 7VV4, 7VV5, 7VDH and 7VV6, respectively. All other data are available from the corresponding authors on request.

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Acknowledgements

We thank the staff at the Core Facilities at the School of Life Sciences, Peking University for help with negative staining EM; the staff at the Cryo-EM Platform and the Electron Microscopy Laboratory of Peking University for help with data collection; the staff at the High-performance Computing Platform of Peking University for help with computation; the staff at the National Centre for Protein Sciences at Peking University for technical assistance; and the staff at the cryo-EM facilities of Southern University of Science and Technology. This work was supported by the National Key R&D Program of China (2018YFC1003600 to X. Yu and J.-P.S.; 2019YFA0904200 to J.-P.S. and P.X.; and 2019YFA0508904 to N.G.), the National Natural Science Foundation of China (91939301 to J.-P.S.; 92057121 to X. Yu; 31900936 to F.Y.; 31971195 to P.X.), the National Science Fund for Distinguished Young Scholars Grant (81825022 to J.-P.S.; and 31725007 to N.G.), the National Science Fund for Excellent Young Scholars (81822008 to X. Yu and 82122070 to F.Y.), the Major Basic Research Project of Shandong Natural Science Foundation (ZR2020ZD39 to J.-P.S.), the Beijing Natural Science Foundation (Z200019 to J.-P.S.). The scientific calculations in this paper were performed on the HPC Cloud Platform of Shandong University.

Author information

Authors and Affiliations

Authors

Contributions

J.-P.S. and N.G. designed and supervised the overall project. J.-P.S., F.Y. and N.G. participated in all data analysis and interpretation. J.-P.S. and N.G. guided all structural analysis. F.Y. and L.G. generated the MRGPRX2 insect cell expression construct, established the C48/80–MRGPRX2–Gi1–scFv16, PAMP-12–MRGPRX2–Gi1–scFv16 and C14linear–MRGPRX2–Gi1–scFV16 complex purification protocol, and prepared samples for the cryo-EM. Y.L. and G.W. prepared the cryo-EM grids, collected the cryo-EM data, and performed the cryo-EM map calculation, model building and refinement. X. Yu, J.W. and L.G. performed the Gi–Gq dissociation assay. C.Z. performed MD simulations. Z.Z. analysed the structure the C14linear–MRGPRX2–Gi1–scFV16 complex. J.W., G.-X.F., J.Z., L.L. and X. Yan generated all of the MRGPRX2 constructs and mutants for the cell-based G-protein activity assays. C.Z. and Y.F.X. performed MD simulations. J.-P.S. and X. Yu designed the cellular functional assays for MRGPRX2. J.-P.S. and X. Yu designed the FlAsH–BRET assays and provided insightful ideas and experimental designs. L.G., J.W., Q.L., Z.L., X.C. and F.Y. designed all of the mutations for determining ligand-binding sites. F.Y., L.G., P.X. and J.W. prepared the figures. J.-P.S. wrote the manuscript. N.G. and X. Yu revised the manuscript. All of the authors have seen and commented on the manuscript.

Corresponding authors

Correspondence to Ning Gao or Jin-Peng Sun.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Hugo Gutierrez-de-Teran and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Purification of MRGPRX2–Gi1–scFv complex.

a-d: To increase receptor expression, thermostabilized cytochrome b562RIL (BRIL) was incorporated at the N-terminus of full-length MRGPRX2, and the chimaeric protein was found to have G protein coupling activity similar to that of wild-type MRGPRX2. Dose response curves of the C48/80 (a), PAMP-12 (b), C14linear (c) and SP (d) induced Gαi–Gγ dissociation in MRGPRX2-WT or Bril-MRGPRX2 overexpressing cells. Data from three independent experiments are presented as the mean ± SEM (n=3). e-h: Representative elution profile of in vitro reconstituted C48/80–MRGPRX2–Gi1 trimer complex (e), PAMP-12–MRGPRX2–Gi1 trimer complex (f), C14linear–MRGPRX2–Gi1 trimer complex (g) and SP–MRGPRX2–Gi1 trimer complex (h) on Superose 6 Increase 10/30 column and SDS-PAGE of the size-exclusion chromatography peak. Representative Figures from three independent experiments were shown.

Extended Data Fig. 2 Cryo-EM images and single particle reconstruction of the C48/80–MRGPRX2–Gi1 complex.

a-b, Cryo-EM micrograph (a) and reference-free two-dimensional class averages of the C48/80–MRGPRX2–Gi1 trimer complex (b). Representative Cryo-EM micrograph from 6562 movies (shown in c) and representative two-dimensional class averages determined using approximately 0.26 million (state 1) or 0.32 million (state 2) particles after 3D classification were shown. c, Flow chart of cryo-EM data processing of C48/80–MRGPRX2–Gi1 complex. d, Fourier shell correlation curves for the final 3D density maps of C48/80- bound MRGPRX2–Gi1 state 1 (Blue) and state 2 (Orange). At the FSC 0.143 cut-off, the overall resolution for state 1 and state 2 are 2.76 Å and 2.90 Å, respectively (left panel). e, 3D density map colored according to local resolution (Å) of the C48/80–MRGPRX2–Gi1 trimer complex.

Extended Data Fig. 3 Electron microscopy density map of C48/80–MRGPRX2–Gi1 and PAMP-12–MRGPRX2-Gi1 complex.

a-d, EM density of the TM helices of MRGPRX2 and the α5 helix of Gαi1 of C 48/80–MRGPRX2–Gi1 (a), PAMP-12–MRGPRX2–Gi1 (b), C14linear–MRGPRX2–Gi1 complex (c) and SP–MRGPRX2–Gi1 complex (d) respectively. All seven-TM bundles were unambiguously traceable in the cryo-EM density map, and the densities of large hydrophobic residues were utilized to assign the primary sequence of MRGPRX2. e, Position of ligand-binding pockets in the C14linear–MRGPRX2–Gi1, SP–MRGPRX2–Gi1, β2AR–Gs (PDB: 3SN6), CB1–Gi (PDB: 6N4B), μ-opioid–Gi (PDB: 6DDF) and GPR97–mGo (PDB: 7D76) complex. The distance between ligand and “toggle switch” were shown. f, Dose response curves of the C48/80, PAMP-12, C14linear and SP induced Gαq–Gγ dissociation in MRGPRX2 overexpressing cells. Data from three independent experiments are presented as the mean ± SEM (n=3). All data were analysed by two-sided one-way ANOVA with Turkey test.

Extended Data Fig. 4 Effects of mutations in the ligand-binding pocket of MRGPRX2 on C48/80 induced dissociation of Gαi–Gγ or Gαq–Gγ.

a, Key interactions between the ethylamine group of the C48/80 and W2436.55, F170ECL2, C1684.64, C1805.34, W2486.60 of the MRGPRX2. The disulfide bond between the C168 and C180 was highlight in cyan. b, Key interactions between the amide of the ethylamine group of C48/80 and E164, D184 of MRGPRX2. Hydrogen bonds were showed in red dash. c, Important residues in MRGPRX2 that recognize the upper “Y” arm of C48/80. d, Elisa experiments to determine the expression levels of the MRGPRX2 wild type and corresponding mutants. Values are mean ± SEM from three independent experiments (n=3). n.s., no significance. All data were analysed by two-sided one-way ANOVA with Turkey test. e, Effects of mutations in the ligand-binding pocket residues of MRGPRX2 on C48/80–induced Gαi–Gγ dissociation in MRGPRX2 overexpressing HEK293 cells. Natural occurred SNPs were highlight in blue. ND, not detectable due to low signal. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). All data were determined by two-sided one-way ANOVA with Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (P =0.0028, 0.0001, 0.0171,  < 0.0001, 0.0045, ND,  < 0.0001,  < 0.0001, ND, 0.0224, 0.0178,  < 0.0001, 0.0050,  < 0.0001, 0.0008, 0.0007 from top to bottom.) f, Effects of mutations of key residues of MRGPRX2 on C48/80–induced Gαq–Gγ dissociation in MRGPRX2 overexpressing HEK293 cells. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3) except E164A and D254A which are the mean ± SEM of five independent experiments (n=5). All data were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001. (P < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001 from top to bottom).

Extended Data Fig. 5 FlAsH insertion site screening and FlAsH BRET experiments.

a, Schematic representation of the FlAsH-BRET assay. The Nluc was inserted at the N-terminal of wild-type MRGPRX2 and the FlAsH motifs were inserted at different positions of extracellular loops of the MRGPRX2. b, Detailed description of the FlAsH motif insertion sites at the extracellular loops of MRGPRX2. FlAsH motifs are labeled in red. c, Elisa experiments to determine the expression levels of the wild-type MRGPRX2 and FlAsH motif incorporated MRGRX2 mutants. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). n.s., no significance. All data were analysed by two-sided one-way ANOVA with Turkey test. d, Representative dose response curves of the conformational changes in response to binding of C48/80 reported by different MRGPRX2 FlAsH-BRET sensors. Data from three independent experiments are presented as the mean ± SEM (n=3). All data were analysed by two-sided one-way ANOVA with Turkey test. e, Elisa experiments to determine the expression levels of the wild type and corresponding mutants of MRGPRX2 using FlAsH-BRET sensor-3 construct. Data from three independent experiments are presented as the mean ± SEM (n=3). n.s., no significance. All data were analysed by two-sided one-way ANOVA with Turkey test. f, Representative dose response curve of the C48/80 induced BRET ratio in HEK 293 cell over-expressing FlAsH-BRET sensor-3 of MRGPRX2 (including wild type or corresponding mutants). Data from three independent experiments are presented as the mean ± SEM (n=3). g, Effects of mutations of the ligand-binding pocket residues of MRGPRX2 on changes in ΔpEC50 in response to stimulation of C48/80 or different polycationic compounds, evaluated using a FlAsH-BRET assay. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). All data were determined by two-sided one-way ANOVA with Tukey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (From top to bottom, C48/80 panel: P < 0.0001,  < 0.0001, 0.7568,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.4929,  < 0.0001,  < 0.0001, 0.0003,  < 0.0001. Atracurium panel: P < 0.0001, 0.0003, 0.0008,  < 0.0001,  < 0.0001, 0.001. Tubocurarine panel: P=0.0206, 0.011, 0.0005, 0.0064, 0.0011, 0.0017.  Ciprofloxacin panel: P < 0.0001, 0.0008,  < 0.0001, ND, ND,  < 0.0001. Rocuronium panel: P =0.0423, 0.0024,  < 0.0001, 0.0003,  < 0.0001, 0.0008). h, Structures of the representative polycationic compounds used in this study. i, Sequence alignment of the MRGPRX2 residues responsible for specific interaction with C48/80 with the corresponding sequences of other MRGPR members. The key residues involved in the binding pocket are colored in red.

Extended Data Fig. 6 Effects of mutations of key residues of MRGPRX2 on polycationic compound drugs induced MRGPRX2 activation.

a, Effects of mutations of key residues of MRGPRX2 on polycationic compound drugs induced MRGPRX2 activation via Gαi–Gγ dissociation assay. The SNP mutations were highlighted in blue. ND, not detectable due to low signal. Data from three independent experiments are presented as the mean ± SEM (n=3). (Atracurium: P =0.0018, 0.0013, ND, ND,  < 0.0001, ND, 0.0064, ND from top to bottom. Tubocurarine: P =0.0003, 0.0013, 0.0037, ND, 0.0064,  < 0.0001,  < 0.0001, ND from top to bottom. Ciprofloxacin: P < 0.0001, 0.0003, ND, 0.0006, ND, ND, 0.0267, 0.0012 from top to bottom. Rocuronium: P =0.0410, 0.0132, ND, ND, ND, 0.0078, 0.9383, ND from top to bottom). b, Effects of mutations of key residues of MRGPRX2 on polycationic compound drugs induced MRGPRX2 activation via Gαq–Gγ dissociation assay. The SNPs were highlight in blue. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3) except values of E164A and W243R in the ligand Ciprofloxacin, values of F170A, D184A, D184H, W243A and W243R in the ligand Rocuronium are the mean ± SEM of five independent experiments (n=5). (Atracurium: P < 0.0001,  < 0.0001, 0.0181, 0.0010, 0.0013,  < 0.0001,  < 0.0001,  < 0.0001 from top to bottom. Tubocurarine: P < 0.0001,  < 0.0001, 0.0002, 0.0021,  < 0.0001, 0.0056, 0.0004,  < 0.0001 from top to bottom. Ciprofloxacin: P < 0.0001, 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0007,  < 0.0001 from top to bottom. Rocuronium: P < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0020,  < 0.0001,  < 0.0001,  < 0.0001). a-b, Upper panel, bar graph representation of pEC50. Lower panel, dose response curves. Statistical differences between WT and mutations were determined by two-sided one-way ANOVA with Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference.

Extended Data Fig. 7 The mode of interaction between peptidic allergens and MRGPRX2.

a: The EM density corresponding to PAMP-12 and potential interaction residues in MRGPRX2. In the EM density map of the PAMP-12–MRGPRX2–Gi complex, a potential density corresponding to PAMP-12 was identified. PAMP-12 lay flat and parallel to the plasma membrane, flanked by TM2-TM3 and ECL1 on one side and TM4-TM5 and ECL3 on the other side. b: The EM density corresponding to C14linear and potential interaction residues in MRGPRX2. c: Alanine scanning mutagenesis and subsequent ligand-binding assays identified key residues in MRGPRX2 that recognize PAMP-12. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). Statistical differences between WT and mutant strains were determined by two-sided one-way ANOVA with Tukey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (P=0.0003, 0.0805, 0.0757, 0.0053, 0.4707, 0.4720, 0.0009, 0.0208, 0.0049, 0.0002, 0.0001 from top to bottom). d, Effects of different PAMP-12 mutations on PAMP-12 binding. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3) except W13A and R20 A which are the mean ± SEM of five independent experiments (n=5). e, Effects of different PAMP-12 mutations on PAMP-12 induced Gαi–Gγ dissociation in MRGPRX2 overexpressing HEK293 cells. Bar graph for EC50 was presented. The EC50 with five-fold change vs. WT was indicated by a dotted blue line. Statistical differences between PAMP-12 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001. The curve data from three independent measurements are measured as mean ± SEM (n=3). (P=0.0004,  < 0.0001,  < 0.0001, 0.0057,  < 0.0001, 0.0004,  < 0.0001,  < 0.0001, 0.0022, 0.0015,  < 0.0001 from left to right). f, Alanine scanning mutagenesis and subsequent ligand-binding assays of the key residues in MRGPRX2 that recognize PAMP-12. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3) except the T106A and F257A which are the mean ± SEM of five independent experiments (n=5). Statistical differences between WT and mutant strains were determined by two-sided one-way ANOVA with Tukey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (P < 0.0001, 0.0004,  < 0.0001, 0.0016, 0.0024,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0005, 0.0002, 0.0757, 0.0010, 0.2988 from top to bottom) g, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on PAMP-12 induced Gαi–Gγ dissociation in MRGPRX2 overexpressing cells. Statistical differences between MRGPRX2 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. The curve data from at least three independent measurements are measured as mean ± SEM (n=3). (P=0.0014, 0.0004, 0.0004, 0.0012,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0004, 0.0004, 0.4361, 0.0401, 0.2415 from top to bottom).

Extended Data Fig. 8 Binding of PAMP-12 to the MRGPRX2.

a, Pairing of PAMP-12 mutants with MRGPRX2 mutants to identify hot spot interactions. We compared the binding capacity of PAMP-12 mutants with that of the PAMP-12 WT for MRGPRX2 mutations with significant deficiency in Gαi–Gγ dissociation. For a receptor mutation that had impaired response to PAMP-12 WT but show little deteriorated response to an PAMP-12 with a selective alanine substitution, the receptor mutated site and ligand mutated site could be paired with each other to identify potential hot spot interactions (highlight in red). Data are from three independent experiments. The original data of Figure 3a. b, The EM density corresponding to five residues from F9 to W13 of PAMP-12 and their interaction residues in ligand-binding pocket of MRGPRX2. EM density corresponding to PAMP-12 were highlight in orange. c, Structural representation of seven representative clusters derived by covalent docking of seven residues of PAMP-12 using Autodock4. d: Docking clusters were ranked by numbers in each cluster. The representative conformations in the classification with either the largest number (cluster 1) and the lowest binding energy (cluster 2) were used for MD simulations, respectively. e, The average RMSD value of the seven residues N14-R20 (N14, K15, W16, A17, L18, S19, R20) in PAMP-12 (upper panel) and RMSD of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) during triplicate 200 ns MD simulations. f-h, Structural representations of the interactions surrounding the R10 (g), W13 (h), F9 and W16 (i) of PAMP-12 of MRGPRX2. Hydrogen bonds were showed in red dash. i, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on PAMP-12 induced Gαq–Gγ dissociation in MRGPRX2 overexpressing cells. Statistical differences between MRGPRX2 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001. The curve data from at least three independent measurements are measured as mean ± SEM (n=3). (P < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0007,  < 0.0001,  < 0.0001 from top to bottom).

Extended Data Fig. 9 Binding of C14linear to the MRGPRX2.

a, Heatmap of pairing of C14linear mutants with MRGPRX2 WT and MRGPRX2 alanine scanning mutants. The receptor mutants that did not show significantly decreased EC50 values compared to those of the WT receptor when binding to a specific C14linear mutant are highlighted by red color. Biochemical characterization indicated that P1 and K3 of C14linear paired with the hydrophobic residue F170ECL2 and the negatively charged residues E1644.60 and D1845.38 of MRGPRX2, respectively. Whereas F5 of C14linear interacted with F170ECL2 and F2577.31 of MRGPRX2, the F6 mutation of C14linear functionally coupled with the F2396.51 and W2436.55 mutations. b, Effects of different C14linear mutations on C14linear induced Gαi–Gγ dissociation in MRGPRX2 overexpressing HEK293 cells. Statistical differences between C14linear WT and mutations were determined by two-sided one-way ANOVA with Tukey test. P < 0.01; ***, P < 0.001, n.s., no significant difference. Data are from three independent experiments. (n=3). (P < 0.0001,  < 0.0001, 0.0557, 0.0011,  < 0.0001,  < 0.0001, 0.0199, 0.0036, 0.0006, 0.0074, 0.0071,  < 0.0001 from left to right). c, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on C14linear induced Gαi–Gγ dissociation in MRGPRX2 overexpressing cells. Statistical differences between MRGPRX2 WT and mutations were determined by two-sided one-way ANOVA with Tukey test. **, P < 0.01; ***, P < 0.001, n.s., no significant difference. Data are from three independent experiments (n=3). (P=0.0023, 0.0005, 0.0002,  < 0.0001, 0.0002,  < 0.0001, 0.0034, 0.0029, 0.0450, 0.0168 from left to right). d, Pairing of C14linear mutants with MRGPRX2 mutants to identify hot spot interactions. We compared the binding capacity of C14linear mutants with that of the C14linear WT for MRGPRX2 mutations with significant deficiency in Gαi–Gγ dissociation. For a receptor mutation that had impaired response to C14linear WT but show little deteriorated response to an C14linear with a selective alanine substitution, the receptor mutated site and ligand mutated site could be paired with each other to identify potential hot spot interactions (highlight in red). Data are from three independent experiments. e-f, Structural representation and EM density of C14linear peptide (e) and C14circular peptide (f). Because the C14linear–MRGPRX2–Gi complex had higher resolution, unbiased alanine scanning mutagenesis of each residue in C14linear and the pocket residues of MRGPRX2 was exploited to assign the primary sequence of C14linear in the EM density. g, Structural model of the interactions between the P1-W7 segment of the C14linear peptide and MRGPRX2, which were derived by computational simulation. h, RMSD of the seven residues P1-W7 in C14linear (upper panel) and of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) during triplicate 200 ns MD simulations. i-j, The average RMSD and +/- s.e.m. values of the seven residues P1-W7 in C14linear (upper panel) and of key residues in MRGPRX2 which directly interact with the seven residues (lower panel) from triplicate 200 ns MD simulations. k, Structural representation and EM density corresponding to S11, S12, C13 and K14 of C14linear.

Extended Data Fig. 10 Binding of SP and Consensus peptidic motif recognized by MRGPRX2.

a, Structural representation and EM density corresponding to four residues from R1 to P4 of SP. The EM density of the SP–MRGPRX2–Gi complex structure enabled us to position the main chain of the first four residues of SP, which assumed different configurations compared to PAMP-12 and C14linear, likely due to the presence of the two prolines in the SP sequence. There was continuous EM density following the first four residues. b, The average RMSD value of the seven residues R1-F7 in SP (upper panel) and of key residues in MRGPRX2 that directly interact with the seven residues (lower panel) during triplicate 200 ns of MD simulation. c, Effects of different mutations within the ligand-binding pocket of MRGPRX2 on SP-induced Gαi–Gγ dissociation in MRGPRX2-overexpressing cells. Data from three independent experiments are presented as the mean ± SEM (n=3). (P=0.0007, 0.0012, 0.0003, 0.0041,  < 0.0001, ND, 0.0002, ND, 0.0003 from top to bottom). d, Interaction differences in the pocket of the MRGPRX2 bound by C48/80, PAMP-12 and C14linear and SP. Residues of MRGPRX2 that interact with ligands are indicated by cyan circle. Residues of MRGPRX2 showing no interaction with C48/80 or PAMP-12 are indicated by blank circle. Residues of MRGPRX2 that interact with simulated parts of ligands and verified by unbiased alanine scanning are indicated by green. e, The binding energy contribution of residues in PAMP-12 as calculated by the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method. f, Mutations of PAMP-12, C14linear, Sermorelin, Exenatide and Enfuvirtide used in Figure 2e. g, Effects of the φp9(X0-1) R/Kp10(X2) φp13(X2-3) φp16(X3) R/Kp20 motif mutations on PAMP-12-, C14linear induced MRGPRX2 activation evaluated via Gαi–Gγ dissociation assay. Data from three independent experiments are presented as the mean ± SEM (n=3). All data were analysed by two-sided one-way ANOVA with Turkey test. h, Effects of mutations of key residues recognizing the φp9(X0-1) R/Kp10(X2) φp13(X2-3) φp16(X3) R/Kp20 motif on MRGPRX2 activation in response to PAMP-12, cortistatin-14, sermorelin, enfuvirtide or exenatide evaluated via Gαi–Gγ dissociation assay. (From top to bottom, for PAMP-12 panel: P=0.0004, 0.0004, 0.0012,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001; C14linear panel: 0.0023, 0.0013, 0.0020, 0.0005, 0.0002,  < 0.0001,  < 0.0001; sermorelin panel: 0.0007, ND,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0004; enfuvirtide panel: ND, ND, 0.0001,  < 0.0001,  < 0.0001, 0.1193,  < 0.0001; exenatide panel: 0.0075, ND, ND,  < 0.0001, ND, 0.0010, 0.0010).

Extended Data Fig. 11 Active state and G protein coupling of MRGPRX2.

a, Structural comparison of TM3 and TM6 in the MRGPRX2–Gi1 complex (green), CB1–Gi complex (cyan, PDB: 6N4B) and μ-opioid–Gi complex (blue, PDB: 6N4B). Compared to the corresponding structures in the CB1 and μ-opioid receptors, the upper TM6 of MRGPRX2 is distorted inwardly at the position of G2366.48 in MRGPRX2. b, Structural comparison of residues involved in kink formation in MRGPRX2 (green), CB1 (cyan, PDB: 6N4B) and μ-opioid receptor (blue, PDB: 6N4B). The engagement of the phenolic oxygen of Y1133.36 and the main chain of G2366.48 in MRGPRX2 at the kink position was further stabilized by packing between F2396.51 and Y1133.36. Toggle switch W6.48 of the CB1, ADGRG3 and µ-opioid receptors was replaced by G2366.48 in MRGPRX2. H-bonds are depicted as dashed lines. c, Structural comparison of TM6 in MRGPRX2 (green), CB1 (cyan, PDB: 6N4B), μ-opioid receptor (light blue, PDB: 6N4B), ADGRG3 (yellow orange, PDB: 7D76), GLPR1 (blue, PDB: 6B3J) and GABA1 (grey, PDB: 7C7Q). d, Cutaway view of G6.48XXF6.52 motif in C14linear–MRGPRX2–Gi1 and C48/80–MRGPRX2–Gi1 complex. Hydrogen bonds are depicted as red dashed lines. e, Schematic representation of the FlAsH-BRET assay. The Nluc was inserted at the P102 position close to extracellular end of TM3 and the FlAsH motif were inserted at the K251 position of the extracellular end of TM6 (upper panel). Representative dose response curves of the conformational changes in response to binding of C 48/80 or Compound 2 reported by FlAsH-BRET assay. A future solution of the inactive structure of MRGPRX2 and comparison with our active structure will provide further evidence of whether kink formation of TM6 is required for the activation of MRGPRX2. Data from three independent experiments are presented as the mean ± SEM (n=3) (lower panel). f, Comparison of the D/ERY motif and NPXXY motif in MRGPRX2–Gi (green), CB1–Gi (gray) and μ-opioid (yellow) complex, which showed similar conformations. g, Structural representation of the MRGPRX2–Gi1 complex (green). TM3-TM5 and ICL1-ICL3 of MRGPRX2 and the αN-helix, α5-helix, i3 loop, and β2-β3 loop of Gαi are highlighted. h, Comparison of the Gi coupling interfaces in cryo-EM structures of MRGPRX2–Gi1 complexes, the μ-opioid–Gi complex (PDB: 6N4B), the CB1–Gi complex (PDB: 6N4B), and the model of MRGPRX2–Gαq generated by computational simulation. Residues of MRGPRX2, μ-opioid receptor or CB1 in contact with Gi are illustrated as green or blue dots. The basic charged R that contributes to the interface is highlighted in blue. Residues that contact Gαi but not Gαq are illustrated as brown dots. i, Detailed interactions between ICL1 and ICL3 of MRGPRX2 and the Gi1 trimer. R60ICL1 inserted in a hydrophobic pocket created by L55 and F335 and formed a hydrogen bond with S334 of the Gβ1 subunit, R214 ICL3 formed a cation-π interaction with Y320 and potential charge interactions with E318 of Gαi1.

Extended Data Fig. 12 MRGPRX2 interactions with Gi and Gq.

a, Effects of mutations in the MRGPRX2 along the Gi trimer interface on PAMP-12 induced Gαi–Gγ dissociation. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). Statistical differences between WT and mutations were determined by two-sided one-way ANOVA with Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (P  < 0.0001,  < 0.0001, 0.0022, 0.0018,  < 0.0001, 0.0008,  < 0.0001, 0.0395, 0.0009, 0.0009, 0.0003, 0.0006, 0.0010, 0.0178, 0.0018, 0.0003, 0.0003 from from top to bottom). b, RMSD of PAMP-12–MRGPRX2 (upper panel) and Gαi (lower panel) during 200 ns MD simulations. c,RMSD of PAMP-12–MRGPRX2 (upper panel) and Gαq (lower panel) during 200 ns MD simulations. d, PAMP-12–MRGPRX2–Gαq complex model after 200 ns MD simulation using M1R-Gq/G11 complex as the Gq template (PDB: 6OIL). e, Structural comparison of PAMP-12–MRGPRX2–Gαq stimulated model (orange) and PAMP-12–MRGPRX2–Gi complex (green). f, Effects of mutations in the MRGPRX2 along the Gαq trimer interface on PAMP-12 induced Gαq–Gγ protein dissociation. Values are the mean ± SEM of three independent experiments for the wild type (WT) and mutants (n=3). Statistical differences between WT and mutations were determined by two-sided one-way ANOVA with Tukey test. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., no significant difference. (P < 0.0001, 0.0016,  < 0.0001,  < 0.0001, 0.9226, 0.8226, 0.8815,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001,  < 0.0001, 0.0319 from top to bottom). g-i, Because the OD1 and OD2 in the carboxylate group of aspartic acid (Asp), or OE1 and OE2 in the carboxylate group of glutamic acid (Glu), or the NH1 and NH2 in guanidinium group of arginine (Arg) are equal atoms respectively, we named the ODc:NHC pair as closer pair for the contacting Asp: Arg, OEC:NHC pair as closer pair for the contacting Glu: Arg; and the remote ODR atom, OER atom, or NHR atom was named in Asp, Glu or Arg respectively g, The average distances between NHC in R140 of MRGPRX2 and oxygen atom of N198 of Gαq in MRGPRX2–Gαq complex or nitrogen atom of the guanidium group of R140 (NE) of MRGPRX2 and ODC of D193 of Gαi1 in MRGPRX2–Gi1 complex calculated from triplicate 200 ns MD simulations. h, The average distances between NHC in R143 of MRGPRX2 and NHC in R24 of Gαq in MRGPRX2–Gαq complex or NHC in R143 of MRGPRX2 and OEC in E28 of Gαi1 in MRGPRX2–Gαi1 complex calculated from triplicate 200 ns MD simulations. i, The average distances between NHC in R214 of MRGPRX2 and carbon atom of the methyl group I323(CD) in MRGPRX2–Gq complex or NHC in R214 of MRGPRX2 and OEC in E318 of Gαi1 in MRGPRX2–Gαi1 complex calculated from triplicate 200 ns MD simulations. j: The effects of selected SNPs on ligand-induced MRGPRX2 activation. SNPs located in the ligand or Gi protein interface of MRGPRX2 are summarized. The Gαi/Gαq–Gγ dissociation assays were measured in the current study, whereas calcium mobilization and mast cell degranulation were obtained from previous studies20 .

Extended Data Table 1 Cryo-EM data collection, model refinement and validation statistics

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Yang, F., Guo, L., Li, Y. et al. Structure, function and pharmacology of human itch receptor complexes. Nature 600, 164–169 (2021). https://doi.org/10.1038/s41586-021-04077-y

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