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- PDB-9umj: Structure of EP54 bound mouse C3aR in complex with Go -

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Entry
Database: PDB / ID: 9umj
TitleStructure of EP54 bound mouse C3aR in complex with Go
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Antibody fragment ScFv16
  • EP54 Ligand
  • Muscarinic acetylcholine receptor M4,C3a anaphylatoxin chemotactic receptor
KeywordsSIGNALING PROTEIN / GPCR / G protein / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


complement component C3a receptor activity / Peptide ligand-binding receptors / Regulation of Complement cascade / Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / vesicle docking involved in exocytosis ...complement component C3a receptor activity / Peptide ligand-binding receptors / Regulation of Complement cascade / Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / G alpha (i) signalling events / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / mu-type opioid receptor binding / corticotropin-releasing hormone receptor 1 binding / vesicle docking involved in exocytosis / positive regulation of neutrophil chemotaxis / G protein-coupled dopamine receptor signaling pathway / regulation of locomotion / positive regulation of macrophage chemotaxis / regulation of heart contraction / parallel fiber to Purkinje cell synapse / positive regulation of vascular endothelial growth factor production / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / postsynaptic modulation of chemical synaptic transmission / Neutrophil degranulation / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / muscle contraction / locomotory behavior / calcium-mediated signaling / negative regulation of insulin secretion / G protein-coupled receptor activity / GABA-ergic synapse / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / positive regulation of angiogenesis / chemotaxis / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / G protein activity / cell body / presynaptic membrane / GTPase binding / Ca2+ pathway / fibroblast proliferation / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / G alpha (i) signalling events / G alpha (s) signalling events / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / chemical synaptic transmission / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / postsynaptic membrane / cell surface receptor signaling pathway / Extra-nuclear estrogen signaling / cell population proliferation / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / dendrite / GTP binding / protein-containing complex binding / glutamatergic synapse / signal transduction / extracellular exosome
Similarity search - Function
C3a anaphylatoxin chemotactic receptor / Muscarinic acetylcholine receptor M4 / Anaphylatoxin chemotactic receptor, C3a/C5a1/C5a2 / Muscarinic acetylcholine receptor family / Formyl peptide receptor-related / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit ...C3a anaphylatoxin chemotactic receptor / Muscarinic acetylcholine receptor M4 / Anaphylatoxin chemotactic receptor, C3a/C5a1/C5a2 / Muscarinic acetylcholine receptor family / Formyl peptide receptor-related / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
C3a anaphylatoxin chemotactic receptor / Muscarinic acetylcholine receptor M4 / Guanine nucleotide-binding protein G(o) subunit alpha / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsBanerjee, R. / Yadav, M.K. / Yadav, R. / Ganguly, M. / Mishra, S. / Dalal, A. / Gati, C. / Shukla, A.K.
Funding support India, United Kingdom, 3items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB)IPA/2020/000405 India
Wellcome TrustIA/S/20/1/504916 United Kingdom
Science and Engineering Research Board (SERB)CRG/2022/002646 India
CitationJournal: bioRxiv / Year: 2025
Title: Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors.
Authors: Sudha Mishra / Manish K Yadav / Annu Dalal / Manisankar Ganguly / Ravi Yadav / Kazuhiro Sawada / Divyanshu Tiwari / Nabarun Roy / Nilanjana Banerjee / Jenny N Fung / Jianina Marallag / ...Authors: Sudha Mishra / Manish K Yadav / Annu Dalal / Manisankar Ganguly / Ravi Yadav / Kazuhiro Sawada / Divyanshu Tiwari / Nabarun Roy / Nilanjana Banerjee / Jenny N Fung / Jianina Marallag / Cedric S Cui / Xaria X Li / John D Lee / Calvin Aaron Dsouza / Shirsha Saha / Parishmita Sarma / Ganita Rawat / Houming Zhu / Htet A Khant / Richard J Clark / Fumiya K Sano / Ramanuj Banerjee / Trent M Woodruff / Osamu Nureki / Cornelius Gati / Arun K Shukla /
Abstract: Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and ...Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on βarr binding and activation using cryo-EM structures. Interestingly, we observe that βarrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.
History
DepositionApr 22, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: EP54 Ligand
C: Muscarinic acetylcholine receptor M4,C3a anaphylatoxin chemotactic receptor
A: Guanine nucleotide-binding protein G(o) subunit alpha
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
H: Antibody fragment ScFv16


Theoretical massNumber of molelcules
Total (without water)160,6246
Polymers160,6246
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules ABG

#3: Protein Guanine nucleotide-binding protein G(o) subunit alpha


Mass: 27024.762 Da / Num. of mol.: 1 / Mutation: G42D,E43N,A227D,G230D,I332A,V335I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAO1 / Production host: Escherichia coli (E. coli)
References: UniProt: P09471, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 38534.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#5: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein/peptide / Protein / Antibody , 3 types, 3 molecules DCH

#1: Protein/peptide EP54 Ligand


Mass: 1211.475 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: Protein Muscarinic acetylcholine receptor M4,C3a anaphylatoxin chemotactic receptor / C3AR / C3a-R / Complement component 3a receptor 1


Mass: 59526.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Mus musculus (house mouse)
Gene: CHRM4, C3ar1, C3r1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08173, UniProt: O09047
#6: Antibody Antibody fragment ScFv16


Mass: 26466.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSourceDetails
1EP54 bound to mouse C3aR in complex with GoCOMPLEXall0MULTIPLE SOURCES
2EP54 ligandCOMPLEX#11SYNTHETIC
3Guanine nucleotide-binding protein G(o) subunit alphaCOMPLEX#31RECOMBINANTThis is a variant of Guanine nucleotide-binding protein G(o) subunit alpha called the "mini G(o) alpha"
4Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1COMPLEX#41RECOMBINANT
5C3a anaphylatoxin chemotactic receptor, C3aR (mouse)COMPLEX#21RECOMBINANT
6Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#51RECOMBINANT
7Antibody fragmentCOMPLEX#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
13Homo sapiens (human)9606
24Homo sapiens (human)9606
35Mus musculus (house mouse)10090
46Homo sapiens (human)9606
57Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Escherichia coli (E. coli)562
24Spodoptera frugiperda (fall armyworm)7108
35Spodoptera frugiperda (fall armyworm)7108
46Spodoptera frugiperda (fall armyworm)7108
57Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.2particle selection
2EPUimage acquisition
4cryoSPARC4.6.2CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC4.6.2initial Euler assignment
11cryoSPARC4.6.2final Euler assignment
12cryoSPARC4.6.2classification
13cryoSPARC4.6.23D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199691 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038341
ELECTRON MICROSCOPYf_angle_d0.4811384
ELECTRON MICROSCOPYf_dihedral_angle_d3.9531185
ELECTRON MICROSCOPYf_chiral_restr0.0411338
ELECTRON MICROSCOPYf_plane_restr0.0031448

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