Abstract
The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.
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The 3D cryo-EM density maps of apo-C3aR–Gi–scFv16, C3a-bound C3aR–Gi–scFv16 and C5a-bound C5aR1–Gi structures have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-34843, EMD-34842 and EMD-34846, respectively. Atomic coordinates for the atomic models of apo-C3aR–Gi–scFv16, C3a-bound C3aR–Gi–scFv16 and C5a-bound C5aR1–Gi structures have been deposited in the Protein Data Bank under the accession numbers 8HK3, 8HK2 and 8HK5, respectively. All relevant data in this paper are included in the manuscript or the Supplementary Information. Source data are provided with this paper.
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Acknowledgements
The cryo-EM data were collected at the Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica (SIMM). We thank all staff at the institution for their assistance in cryo-EM data collection. This work was partially supported by grants from the Special Research Assistant Project of the Chinese Academy of Sciences (to Y.Z.); the Sailing Program of Shanghai Venus Project (grant no. 23YF1456700 to Y.Z.); the Youth Innovation Promotion Association of the Chinese Academy of Sciences (grant no. 2023298 to Y.Z.); the Natural Science Foundation of Shanghai, China (grant no. 23ZR1475300 to Y.Z.); the CAS Strategic Priority Research Program (grant no. XDB37030103 to H.E.X.); the Shanghai Municipal Science and Technology Major Project (grant no. 2019SHZDZX02 to H.E.X.); the Shanghai Municipal Science and Technology Major Project (H.E.X.); the National Natural Science Foundation of China (grant no. 32130022 to H.E.X., grant no. 82121005 to H.E.X. and Y.J., grant no. 32171187 to Y.J.); and the National Key R&D Program of China (grant no. 2018YFA0507002 to H.E.X.).
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Y.W. designed the expression constructs of C3aR and C5aR1, performed data acquisition and structure determination of C5a–C5aR1–Gi–scFv16, performed all functional assays and participated in figure preparation and manuscript editing. W.L. optimized the purification conditions of protein complexes and prepared protein samples of apo-C3aR–Gi–scFv16, C3a–C3aR–Gi–scFv16 and C5a–C5aR-Gi complexes for cryo-EM grid making and data collection and participated in method preparation. Y.Z. performed data acquisition and structure determination of apo and C3a-bound C3aR–Gi–scFv16 complex. Y.X., Q.Y. and Y.Z. built the models and refined the structures. X.H. performed the molecular dynamic simulation and calculation of binding free energy values. P.L., W.F., J.Z. and X.Z. assisted in cloning construction and protein sample preparation. X.C. supervised X.H. in the computational analysis. Y.J. supervised Y.W. and W.L. Y.Z. and H.E.X. conceived and supervised the project and wrote the manuscript. Y.Z. prepared the draft of the manuscript with input from Y.W. and W.L.
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Extended data
Extended Data Fig. 1 Biochemical results of complement system in this study.
a, Schematic representation of the C3aR/C5aR1 and C3a/C5a constructs. b, SDS-PAGE analysis of the recombinant C3a/C5a mutants and truncations. c, Comparisons of the capabilities of homemade C3a and C5a in C3aR and C5aR1 activation using the commercially available C3a (upper panel, Bio-Techne, catalog number: #3677-C3-025) and C5a (lower panel, Acro, catalog number: #P01031) as reference ligands. Data shown are means ± S.E.M. from N = three independent experiments performed in technical triplicate. d, e, f, Size exclusion chromatography profiles (left) and SDS-PAGE analysis (right) of the apo-C3aR–Gi complex (d), C3a–C3aR–Gi complex (e) and C5a–C5aR1–Gi complex (f).
Extended Data Fig. 2 Structure determination of the apo/C3a–C3aR–Gi, and C5a–C5aR1–Gi complex.
a, Representative cryo-EM raw image and 2D classification averages of the apo-C3aR–Gi complex. b, Cryo-EM data processing flowchart of the apo-C3aR–Gi complex. c, The Fourier shell correlation (FSC) curves of the apo-C3aR–Gi complex. The global resolution of the final processed density map estimated at the FSC = 0.143 is 3.2 Å. d, Local resolution and angle distribution map of the apo-C3aR–Gi complex. The density map is shown at 0.08 threshold. e, Representative cryo-EM image and 2D classification averages of the C3a–C3aR–Gi complex. f, Cryo-EM data processing work-flow of the C3a–C3aR–Gi complex. g, The Fourier shell correlation (FSC) curves of the apo-C3aR–Gi complex. The global resolution of the final processed density map estimated at the FSC = 0.143 is 2.9 Å. h, Local resolution and angle distribution map of the C5a–C5aR1–Gi complex. The density map is shown at 0.25 threshold. i, Representative cryo-EM image and 2D classification averages of the C5a–C5aR1–Gi complex. j, Cryo-EM data processing flowcharts of the C5a–C5aR1–Gi complex. k, The Fourier shell correlation (FSC) curves of the C5a–C5aR1–Gi complex. The global resolution of the final processed density map estimated at the FSC = 0.143 is 3.0 Å. l, Local resolution and angle distribution map of the C5a–C5aR1–Gi complex. The density map is shown at 0.11 threshold.
Extended Data Fig. 3 Local electron densities of C3aR–Gi and C5aR1–Gi complexes.
a, b, c, EM density maps of transmembrane helices TM1-TM7 and helix 8 of C3aR or C5aR1, αN or α5 helices of Gi, and ligands C3a and C5a in the apo-C3aR–Gi complex (a), the C3a–C3aR–Gi complex (b), and the C5a–C5aR1–Gi complex(c). The density maps were shown at the thresholds of 0.08, 0.15 and 0.08 for apo-C3aR–Gi complex, the C3a–C3aR–Gi complex, and the C5a–C5aR1–Gi complex, respectively.
Extended Data Fig. 4 Molecular dynamic simulations of C3a and C5a binding poses.
a, Superposition of C3a structure determined by cryo-EM in this study and crystal structure of C3a (PDB: 4HW5). b, Superposition of C5a structure determined by cryo-EM in this study and crystal structure of C5a (PDB: 5B4P). c, Molecular dynamics simulations of C3a and C5a bound to C3aR and C5aR1, respectively.
Extended Data Fig. 5 The effects of tyrosine sulfation of C3aR and C5aR1 in mammalian and insect cell systems.
a, the activation of C3a (left panel) and C5a (right panel) on wild-type or mutant receptors in HEK293 cells. Data shown are means ± S.E.M. from N = three independent experiments performed in technical duplicate. b, the binding profile of C3a (left panel) and C5a (right panel) on wild-type or mutant receptors in Sf9 insect cells. The negative control is receptor without ligands, the purified rhodopsin–GRK1 complex with Flag epitope at rhodopsin N-terminus and His8 tag at GRK1 C-terminus was used as positive control. The data presented represent the mean ± S.E.M. from N = 5 (C3aR) and N = 3 (C5aR1) independent experiments performed in technical triplicate and normalized to wild-type C3aR/C5aR1 with C3a/C5a, respectively. For C3aR, two-tailed Student’s t-test was used for testing statistical significance. For C5aR1, One-way ANOVA with Tukey’s test was used for testing statistical significance. *P < 0.05; **P < 0.01 and ***P < 0.001 were considered statistically significant. The P value of Y174F compared to WT of C3aR is 0.03. The P value of Y11F, Y14F compared to WT of C5aR1 is 0.21 and 0.02, respectively.
Extended Data Fig. 6 The pose and binding free energy of C3a in WT C3aR and C3aR mutants.
a, b, c, Pose of C3a C terminal hook in Y174F C3aR mutant (a), C3aR (b) and C3aR with sulfated Y174 (c). The binding free energy values were shown below each figure.
Extended Data Fig. 7 Conformational changes of C3aR and C5aR1 activation.
a, b, c, Conformational changes upon C3aR activation induced by C3a (top) and C5aR1 activation induced by C5a (bottom) when compared with PMX53-bound inactive C5aR1, including rearrangement of PIF motif (a), alteration of DRF motif (b) and NPxxY motif (c). d, e, f, Conformational changes in the intracellular regions of the receptors when aligned the structures of C3a-bound C3aR with inactive C5aR1 (d), C5a-bound C5aR1 with inactive C5aR1 (e) and C3a-bound C3aR with C5a-bound C5aR1 (f). Lime green, active C3aR; slate, active C5aR1; gray, PMX53-bound inactive C5aR1 (PDB: 6C1R).
Extended Data Fig. 8 Constitutive activity determinants of C3aR.
a, Histogram of constitutive activities of C3aR and C5aR1, pcDNA3.0 vector was used as control. Cells were treated with decreasing dose of fosklin. It can be seen that C3aR has high basal activity whereas C5aR1 has no basal activities and behaves like the control. Data shown are means ± S.E.M. from N = two independent experiments performed in technical duplicate. b, Structural superposition of the C3a–C3aR with the apo-C3aR in orthogonal view (left), extracellular view (middle) and intracellular view (right). Helixes are shown as rods. c, Structural determinants affecting the constitutive activity of C3aR. In apo-C3aR, residue R3405.42 forms direct hydrogen bond with Y3936.51. d, Characterization of the mutational effects of critical residues on the basal activation of C3aR, pcDNA3.0 vector was used as control. Cells were treated with 1 μM fosklin. Data shown are means ± S.E.M. from five independent experiments (n = 5) performed in technical duplicate. Data were analyzed by two-side, one-way ANOVA by Dunnett multiple test compared with WT. *P < 0.05; **P < 0.01 and ***P < 0.001 were considered as statistically significant. P = 0.005, P = 0.08, P < 0.001, P < 0.001 from left to right.
Extended Data Fig. 9 Gi coupling of C3aR and C5aR1.
a, Overall structural superposition of the C3a–C3aR–Gi complex with the C5a–C5aR1–Gi complex. b, Subtle differences of α5 helix of Gαi subunit inserted into C3aR and C5aR1. c, C3a/C5a induced the extracellular region movement between C3aR and C5aR1. d, e, f, Interactions between C3aR and Gαi subunit, (d) intracellular cavity of C3aR with α5 helix of Gαi subunit, (e) ICL2 of C3aR with Gαi subunit, (f) ICL3 of C3aR with Gαi subunit. g, h, i, Interactions between C5aR1 and Gαi subunit, (g) intracellular cavity of C5aR1 with α5 helix of Gαi subunit, (h) ICL2 of C5aR1 with Gαi subunit, (i) ICL3 of C5aR1 with Gαi subunit.
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Unprocessed SDS–PAGE gel for Fig. 1b,d–f.
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Wang, Y., Liu, W., Xu, Y. et al. Revealing the signaling of complement receptors C3aR and C5aR1 by anaphylatoxins. Nat Chem Biol 19, 1351–1360 (2023). https://doi.org/10.1038/s41589-023-01339-w
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DOI: https://doi.org/10.1038/s41589-023-01339-w
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