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- PDB-9kxs: Structure of EP67 bound mouse C5aR1 in complex with Go -

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Entry
Database: PDB / ID: 9kxs
TitleStructure of EP67 bound mouse C5aR1 in complex with Go
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Antibody fragment ScFv16
  • EP67 ligand
  • Muscarinic acetylcholine receptor M4,C5a anaphylatoxin chemotactic receptor 1
KeywordsSIGNALING PROTEIN / GPCR / G protein / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


complement component C5a binding / cell proliferation in hindbrain / response to peptidoglycan / presynapse organization / complement component C5a signaling pathway / Regulation of Complement cascade / Peptide ligand-binding receptors / complement component C5a receptor activity / Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity ...complement component C5a binding / cell proliferation in hindbrain / response to peptidoglycan / presynapse organization / complement component C5a signaling pathway / Regulation of Complement cascade / Peptide ligand-binding receptors / complement component C5a receptor activity / Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / regulation of locomotion / G alpha (i) signalling events / mu-type opioid receptor binding / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / corticotropin-releasing hormone receptor 1 binding / complement receptor mediated signaling pathway / positive regulation of neutrophil chemotaxis / G protein-coupled dopamine receptor signaling pathway / positive regulation of macrophage chemotaxis / parallel fiber to Purkinje cell synapse / amyloid-beta clearance / positive regulation of vascular endothelial growth factor production / negative regulation of insulin secretion / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / postsynaptic modulation of chemical synaptic transmission / neutrophil chemotaxis / muscle contraction / Neutrophil degranulation / astrocyte activation / positive regulation of epithelial cell proliferation / adenylate cyclase-inhibiting serotonin receptor signaling pathway / G protein-coupled serotonin receptor binding / microglial cell activation / mRNA transcription by RNA polymerase II / cognition / GABA-ergic synapse / G protein-coupled receptor activity / positive regulation of angiogenesis / G-protein beta/gamma-subunit complex binding / apical part of cell / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / Prostacyclin signalling through prostacyclin receptor / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G beta:gamma signalling through BTK / photoreceptor disc membrane / ADP signalling through P2Y purinoceptor 12 / Glucagon-type ligand receptors / Sensory perception of sweet, bitter, and umami (glutamate) taste / 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 / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / G alpha (12/13) signalling events / G-protein beta-subunit binding / extracellular vesicle / positive regulation of cytosolic calcium ion concentration / Thrombin signalling through proteinase activated receptors (PARs) / signaling receptor complex adaptor activity / cell body / GTPase binding / presynaptic membrane / cytoplasmic vesicle / G protein activity / chemical synaptic transmission / Ca2+ pathway / negative regulation of neuron apoptotic process / 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 / basolateral plasma membrane / G alpha (q) signalling events / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Ras protein signal transduction / positive regulation of ERK1 and ERK2 cascade / postsynaptic membrane / cell surface receptor signaling pathway / Extra-nuclear estrogen signaling
Similarity search - Function
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 / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit ...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 / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / 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. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 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
Muscarinic acetylcholine receptor M4 / Guanine nucleotide-binding protein G(o) subunit alpha / C5a anaphylatoxin chemotactic receptor 1 / 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: 3.31 Å
AuthorsBanerjee, R. / Yadav, R. / Yadav, M.K. / 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: Proc Natl Acad Sci U S A / Year: 2026
Title: Structural basis of complement anaphylatoxin receptor activation by an immunostimulant lead candidate.
Authors: Annu Dalal / Manish K Yadav / Manisankar Ganguly / Sudha Mishra / Ravi Yadav / Shachie Sinha / Nabarun Roy / Divyanshu Tiwari / Debdatta Mukherjee / Ashna Reyaz / Calvin A Dsouza / Ameesha ...Authors: Annu Dalal / Manish K Yadav / Manisankar Ganguly / Sudha Mishra / Ravi Yadav / Shachie Sinha / Nabarun Roy / Divyanshu Tiwari / Debdatta Mukherjee / Ashna Reyaz / Calvin A Dsouza / Ameesha Nigam / Nilanjana Banerjee / Xaria X Li / Richard J Clark / Trent M Woodruff / Ramanuj Banerjee / Cornelius Gati / Arun K Shukla /
Abstract: Activation of the complement cascade is a primary innate immune response mechanism to combat pathogenic infections. Complement anaphylatoxins (i.e., C3a and C5a) exert a robust inflammatory response ...Activation of the complement cascade is a primary innate immune response mechanism to combat pathogenic infections. Complement anaphylatoxins (i.e., C3a and C5a) exert a robust inflammatory response via prototypical GPCRs (i.e., C3aR and C5aR1). Several peptides derived from anaphylatoxins have shown promise as immunostimulants from therapeutic standpoint by eliciting immune response without excessive inflammation. EP67, a C5a-derived decapeptide, is the most advanced candidate with preclinical indications in antiviral and antibacterial context. Still, the molecular mechanism and the precise receptor target of EP67 remain unclear. Here, we perform a comprehensive pharmacological profiling of EP67 on the human and mouse C3aR and C5aR1 and find that it preferentially activates human C3aR in transducer-coupling assays. Subsequently, we determined four cryo-EM structures of C3aR and C5aR1 in complex with EP67, which elucidate the molecular details of its interaction with, and activation of, these receptors. Interestingly, we observe that EP67 adopts a hook-like structure and binds in the orthosteric pocket of the receptors, analogous to that of the carboxyl terminus of C3a and C5a. We employ site-directed mutagenesis studies to validate the key interactions of EP67 with these receptors and corroborate the structural observations including the engagement of a critical activation switch. Finally, we observe that EP67 induces distinct conformations of the TM7-Helix8 interface for C3aR and C5aR1, which provides a plausible explanation for its ability to preferentially activate C3aR. In summary, our study elucidates molecular insights into the interaction of EP67 with the complement anaphylatoxin receptors, and it should facilitate further optimization for therapeutic applications.
History
DepositionDec 7, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Muscarinic acetylcholine receptor M4,C5a anaphylatoxin chemotactic receptor 1
B: Guanine nucleotide-binding protein G(o) subunit alpha
C: 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
D: EP67 ligand


Theoretical massNumber of molelcules
Total (without water)146,9626
Polymers146,9626
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 BCG

#2: 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
#3: 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
#4: 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 / Antibody / Protein/peptide , 3 types, 3 molecules AHD

#1: Protein Muscarinic acetylcholine receptor M4,C5a anaphylatoxin chemotactic receptor 1 / C5a anaphylatoxin chemotactic receptor / C5a-R / C5aR


Mass: 44958.422 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, C5ar1, C5ar, C5r1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P08173, UniProt: P30993
#5: Antibody Antibody fragment ScFv16


Mass: 27340.482 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)
#6: Protein/peptide EP67 ligand


Mass: 1243.474 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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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 (eV)
1EP67 bound to mouse C5aR1 in complex with GoCOMPLEXall0MULTIPLE SOURCES
2mouse C5a anaphylatoxin chemotactic receptor 1COMPLEX#11RECOMBINANT
3Guanine nucleotide-binding protein G(o) subunit alphaCOMPLEX#21RECOMBINANTThis 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#31RECOMBINANT
5Antibody fragment ScFv16COMPLEX#51RECOMBINANT
6Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2COMPLEX#41RECOMBINANT
7EP67 ligandCOMPLEX#61SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Homo sapiens (human)9606
34Homo sapiens (human)9606
45Mus musculus (house mouse)10090
56Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli (E. coli)562
34Spodoptera frugiperda (fall armyworm)7108
45Escherichia coli (E. coli)562
56Spodoptera frugiperda (fall armyworm)7108
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48.49 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2EPUimage acquisition
4cryoSPARC4.5.3CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.6.2classification
13cryoSPARC4.5.33D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 409104 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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