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- EMDB-41370: Structure of a class A GPCR/Fab complex -

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Basic information

Entry
Database: EMDB / ID: EMD-41370
TitleStructure of a class A GPCR/Fab complex
Map data
Sample
  • Complex: CCR8_Fab complex
    • Protein or peptide: Fab heavy chainFragment antigen-binding
    • Protein or peptide: Fab light chainFragment antigen-binding
    • Protein or peptide: C-C chemokine receptor type 8, Green fluorescent protein fusion
KeywordsGPCR Fab / STRUCTURAL PROTEIN
Function / homology
Function and homology information


chemokine receptor activity / C-C chemokine receptor activity / C-C chemokine binding / Chemokine receptors bind chemokines / coreceptor activity / cell chemotaxis / bioluminescence / generation of precursor metabolites and energy / calcium-mediated signaling / chemotaxis ...chemokine receptor activity / C-C chemokine receptor activity / C-C chemokine binding / Chemokine receptors bind chemokines / coreceptor activity / cell chemotaxis / bioluminescence / generation of precursor metabolites and energy / calcium-mediated signaling / chemotaxis / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / cell adhesion / immune response / G protein-coupled receptor signaling pathway / external side of plasma membrane / plasma membrane
Similarity search - Function
CC chemokine receptor 8 / Chemokine receptor family / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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)
Similarity search - Domain/homology
Green fluorescent protein / C-C chemokine receptor type 8
Similarity search - Component
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit) / Aequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSun D / Johnson M / Masureel M
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.
Authors: Dawei Sun / Yonglian Sun / Eric Janezic / Tricia Zhou / Matthew Johnson / Caleigh Azumaya / Sigrid Noreng / Cecilia Chiu / Akiko Seki / Teresita L Arenzana / John M Nicoludis / Yongchang Shi ...Authors: Dawei Sun / Yonglian Sun / Eric Janezic / Tricia Zhou / Matthew Johnson / Caleigh Azumaya / Sigrid Noreng / Cecilia Chiu / Akiko Seki / Teresita L Arenzana / John M Nicoludis / Yongchang Shi / Baomei Wang / Hoangdung Ho / Prajakta Joshi / Christine Tam / Jian Payandeh / Laëtitia Comps-Agrar / Jianyong Wang / Sascha Rutz / James T Koerber / Matthieu Masureel /
Abstract: The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an ...The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.
History
DepositionJul 27, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41370.png

Downloads & links

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Map

FileDownload / File: emd_41370.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.97 Å
Density
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.47353595 - 1.0154614
Average (Standard dev.)-0.000022880735 (±0.01767573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 271.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41370_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41370_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41370_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : CCR8_Fab complex

EntireName: CCR8_Fab complex
Components
  • Complex: CCR8_Fab complex
    • Protein or peptide: Fab heavy chainFragment antigen-binding
    • Protein or peptide: Fab light chainFragment antigen-binding
    • Protein or peptide: C-C chemokine receptor type 8, Green fluorescent protein fusion

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Supramolecule #1: CCR8_Fab complex

SupramoleculeName: CCR8_Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Fab heavy chain

MacromoleculeName: Fab heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 23.775549 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QSVEESGGRL VTPGTPLTLT CTVSGFSLSN YAMIWVRQAP GEGLEWVGTI SLGGYTYYAN WAKGRFTISK TSTTVDLKIS SPTTEDTAT YFCARARWST DSAIYTYAFD PWGPGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QSVEESGGRL VTPGTPLTLT CTVSGFSLSN YAMIWVRQAP GEGLEWVGTI SLGGYTYYAN WAKGRFTISK TSTTVDLKIS SPTTEDTAT YFCARARWST DSAIYTYAFD PWGPGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD

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Macromolecule #2: Fab light chain

MacromoleculeName: Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 23.402865 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AYDMTQTPAS VEVAVGGTVT IKCQASQSIS SYLSWYQQKP GQRPELLIYK ASTLASGVSS RFKGSGSGTQ FTLTISDLEA ADAATYYCQ QGYTSSNIDN IFGGGTEVVV KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
AYDMTQTPAS VEVAVGGTVT IKCQASQSIS SYLSWYQQKP GQRPELLIYK ASTLASGVSS RFKGSGSGTQ FTLTISDLEA ADAATYYCQ QGYTSSNIDN IFGGGTEVVV KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #3: C-C chemokine receptor type 8, Green fluorescent protein fusion

MacromoleculeName: C-C chemokine receptor type 8, Green fluorescent protein fusion
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 76.003508 KDa
Recombinant expressionOrganism: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
SequenceString: MKTIIALSYI FCLVFADYKD DDDKGSENLY FQSGSMDYTL DLSVTTVTDY YYPDIFSSPC DAELIQTNGK LLLAVFYCLL FVFSLLGNS LVILVLVVCK KLRSITDVYL LNLALSDLLF VFSFPFQTYY LLDQWVFGTV MCKVVSGFYY IGFYSSMFFI T LMSVDRYL ...String:
MKTIIALSYI FCLVFADYKD DDDKGSENLY FQSGSMDYTL DLSVTTVTDY YYPDIFSSPC DAELIQTNGK LLLAVFYCLL FVFSLLGNS LVILVLVVCK KLRSITDVYL LNLALSDLLF VFSFPFQTYY LLDQWVFGTV MCKVVSGFYY IGFYSSMFFI T LMSVDRYL AVVHAVYALK VRTIRMGTTL CLAVWLTAIM ATIPLLVFYQ VASEDGVLQC YSFYNQQTLK WKIFTNFKMN IL GLLIPFT IFMFCYIKIL HQLKRCQNHN KTKAIRLVLI VVIASLLFWV PFNVVLFLTS LHSMHILDGC SISQQLTYAT HVT EIISFT HCCVNPVIYA FVGEKFKKHL SEIFQKSCSQ IFNYLGRQMP RESCEKSSSC QQHSSRSSSV DYILGNSLEV LFQG PMVSK GEELFTGVVP ILVELDGDVN GHKFSVSGEG EGDATYGKLT LKLICTTGKL PVPWPTLVTT LGYGLQCFAR YPDHM KQHD FFKSAMPEGY VQERTIFFKD DGNYKTRAEV KFEGDTLVNR IELKGIDFKE DGNILGHKLE YNYNSHNVYI TADKQK NGI KANFKIRHNI EDGGVQLADH YQQNTPIGDG PVLLPDNHYL SYQSKLSKDP NEKRDHMVLL EFVTAAGITL GMDELYK GS AWSHPQFEKG GGSGGGSGGS AWSHPQFEK

UniProtKB: C-C chemokine receptor type 8, Green fluorescent protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 1.067 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio in cryoSPARC
Initial angle assignmentType: OTHER / Details: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER / Details: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 340479
FSC plot (resolution estimation)

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