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- PDB-9kyu: Structure of beta-arrestin1 in complex with mouse C5aR1pp -

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Basic information

Entry
Database: PDB / ID: 9kyu
TitleStructure of beta-arrestin1 in complex with mouse C5aR1pp
Components
  • Beta-arrestin-1
  • C5aR1 phosphopeptide
  • Fab30 Heavy Chain
  • Fab30 Light Chain
KeywordsSIGNALING PROTEIN / GPCR / G protein / beta-arrestin
Function / homology
Function and homology information


V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / presynapse organization / regulation of inositol trisphosphate biosynthetic process / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding ...V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / presynapse organization / regulation of inositol trisphosphate biosynthetic process / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding / alpha-1B adrenergic receptor binding / Lysosome Vesicle Biogenesis / complement component C5a receptor activity / Regulation of Complement cascade / Peptide ligand-binding receptors / AP-2 adaptor complex binding / response to peptidoglycan / Ub-specific processing proteases / angiotensin receptor binding / MAP2K and MAPK activation / Golgi Associated Vesicle Biogenesis / Cargo recognition for clathrin-mediated endocytosis / clathrin-cargo adaptor activity / G alpha (i) signalling events / Clathrin-mediated endocytosis / negative regulation of interleukin-8 production / regulation of G protein-coupled receptor signaling pathway / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / arrestin family protein binding / G protein-coupled receptor internalization / mitogen-activated protein kinase kinase binding / positive regulation of neutrophil chemotaxis / Thrombin signalling through proteinase activated receptors (PARs) / response to morphine / clathrin binding / stress fiber assembly / positive regulation of Rho protein signal transduction / positive regulation of macrophage chemotaxis / pseudopodium / amyloid-beta clearance / negative regulation of interleukin-6 production / positive regulation of receptor internalization / positive regulation of vascular endothelial growth factor production / negative regulation of Notch signaling pathway / phototransduction / positive regulation of insulin secretion involved in cellular response to glucose stimulus / insulin-like growth factor receptor binding / clathrin-coated pit / neutrophil chemotaxis / Neutrophil degranulation / astrocyte activation / negative regulation of protein ubiquitination / GTPase activator activity / nuclear estrogen receptor binding / positive regulation of protein ubiquitination / positive regulation of epithelial cell proliferation / phosphoprotein binding / mRNA transcription by RNA polymerase II / microglial cell activation / G protein-coupled receptor binding / G protein-coupled receptor activity / negative regulation of ERK1 and ERK2 cascade / cognition / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / endocytosis / apical part of cell / positive regulation of angiogenesis / positive regulation of protein phosphorylation / protein transport / cytoplasmic vesicle / ubiquitin-dependent protein catabolic process / regulation of apoptotic process / basolateral plasma membrane / dendritic spine / molecular adaptor activity / negative regulation of neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / postsynaptic membrane / transmembrane transporter binding / transcription coactivator activity / positive regulation of ERK1 and ERK2 cascade / positive regulation of MAPK cascade / endosome / defense response to Gram-positive bacterium / postsynaptic density / protein ubiquitination / G protein-coupled receptor signaling pathway / response to xenobiotic stimulus / signaling receptor binding / positive regulation of cell population proliferation / ubiquitin protein ligase binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / chromatin / glutamatergic synapse / enzyme binding / positive regulation of transcription by RNA polymerase II / nucleus / plasma membrane
Similarity search - Function
Anaphylatoxin chemotactic receptor, C3a/C5a1/C5a2 / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Formyl peptide receptor-related / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Anaphylatoxin chemotactic receptor, C3a/C5a1/C5a2 / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Formyl peptide receptor-related / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / 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) / Immunoglobulin E-set
Similarity search - Domain/homology
Beta-arrestin-1 / C5a anaphylatoxin chemotactic receptor 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.72 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: 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
DepositionDec 9, 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

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Assembly

Deposited unit
A: Beta-arrestin-1
U: C5aR1 phosphopeptide
I: Fab30 Heavy Chain
M: Fab30 Light Chain
D: Beta-arrestin-1
R: Fab30 Heavy Chain
Q: Fab30 Light Chain
X: C5aR1 phosphopeptide


Theoretical massNumber of molelcules
Total (without water)198,0168
Polymers198,0168
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

#1: Protein Beta-arrestin-1 / Arrestin beta-1


Mass: 47088.508 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Arrb1 / Production host: Escherichia coli (E. coli) / References: UniProt: P29066
#2: Protein/peptide C5aR1 phosphopeptide / C5a anaphylatoxin chemotactic receptor / C5a-R / C5aR / C5a anaphylatoxin chemotactic receptor 1


Mass: 2972.297 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse) / References: UniProt: P30993
#3: Antibody Fab30 Heavy Chain


Mass: 25512.354 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#4: Antibody Fab30 Light Chain


Mass: 23435.064 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
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-IDSource
1beta-arrestin1 in complex with mouse C5aR1ppCOMPLEXall0MULTIPLE SOURCES
2Beta-arrestin-1COMPLEX#11RECOMBINANT
3Fab30 Heavy Chain and Light chainCOMPLEX#3-#41RECOMBINANT
4mouse C5aR1 phosphopeptideCOMPLEX#21SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia 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

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: 3000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 53.7 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
1cryoSPARC4.6.2particle 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: C2 (2 fold cyclic)
3D reconstructionResolution: 2.72 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 376105 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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