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- EMDB-41289: Human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in n... -

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Basic information

Entry
Database: EMDB / ID: EMD-41289
TitleHuman ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in nanodisc
Map data
Sample
  • Complex: human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in nanodisc
    • Protein or peptide: Beta-arrestin-1
    • Protein or peptide: Fab7 heavy chain
    • Protein or peptide: Fab7 light chain
    • Protein or peptide: anti-Fab nanobody
    • Protein or peptide: Atypical chemokine receptor 3
Keywordscomplex / GPCR / arrestin / signaling / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


TGFBR3 regulates TGF-beta signaling / oculomotor nerve development / MAP2K and MAPK activation / Activation of SMO / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / positive regulation of mesenchymal stem cell migration / C-X-C chemokine binding / AP-2 adaptor complex binding / Ub-specific processing proteases ...TGFBR3 regulates TGF-beta signaling / oculomotor nerve development / MAP2K and MAPK activation / Activation of SMO / Golgi Associated Vesicle Biogenesis / Lysosome Vesicle Biogenesis / positive regulation of mesenchymal stem cell migration / C-X-C chemokine binding / AP-2 adaptor complex binding / Ub-specific processing proteases / clathrin coat of coated pit / clathrin heavy chain binding / C-X-C chemokine receptor activity / Cargo recognition for clathrin-mediated endocytosis / desensitization of G protein-coupled receptor signaling pathway / Clathrin-mediated endocytosis / C-C chemokine receptor activity / clathrin-dependent endocytosis / negative regulation of intrinsic apoptotic signaling pathway in response to DNA damage / chemokine-mediated signaling pathway / C-C chemokine binding / G protein-coupled receptor internalization / acetylcholine receptor binding / inositol hexakisphosphate binding / Chemokine receptors bind chemokines / Thrombin signalling through proteinase activated receptors (PARs) / scavenger receptor activity / G alpha (s) signalling events / clathrin binding / small molecule binding / pseudopodium / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / vasculogenesis / coreceptor activity / clathrin-coated pit / cell chemotaxis / G protein-coupled receptor binding / calcium-mediated signaling / recycling endosome / receptor internalization / protein transport / positive regulation of protein phosphorylation / positive regulation of cytosolic calcium ion concentration / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / G alpha (i) signalling events / angiogenesis / molecular adaptor activity / early endosome / positive regulation of ERK1 and ERK2 cascade / endosome / cell adhesion / immune response / external side of plasma membrane / negative regulation of cell population proliferation / intracellular membrane-bounded organelle / cell surface / signal transduction / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Atypical chemokine receptor 3 / : / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Atypical chemokine receptor 3 / : / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / 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 / Atypical chemokine receptor 3
Similarity search - Component
Biological speciesBos taurus (domestic cattle) / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsChen Q / Tesmer JJG
Funding support United States, Denmark, 12 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA254402 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117372 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI161880 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA221289 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA023168 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM137505 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL071818 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA023168 United States
Walther Cancer Foundation United States
Robertson Foundation/Cancer Research InstituteIrvington Postdoctoral Fellowship United States
VILLUM FONDEN00025326 Denmark
Ralph W. and Grace M. Showalter Research Trust4480108 United States
CitationJournal: Nature / Year: 2025
Title: Effect of phosphorylation barcodes on arrestin binding to a chemokine receptor.
Authors: Qiuyan Chen / Christopher T Schafer / Somnath Mukherjee / Kai Wang / Martin Gustavsson / James R Fuller / Katelyn Tepper / Thomas D Lamme / Yasmin Aydin / Parth Agrawal / Genki Terashi / Xin- ...Authors: Qiuyan Chen / Christopher T Schafer / Somnath Mukherjee / Kai Wang / Martin Gustavsson / James R Fuller / Katelyn Tepper / Thomas D Lamme / Yasmin Aydin / Parth Agrawal / Genki Terashi / Xin-Qiu Yao / Daisuke Kihara / Anthony A Kossiakoff / Tracy M Handel / John J G Tesmer /
Abstract: Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote ...Unique phosphorylation 'barcodes' installed in different regions of an active seven-transmembrane receptor by different G-protein-coupled receptor (GPCR) kinases (GRKs) have been proposed to promote distinct cellular outcomes, but it is unclear whether or how arrestins differentially engage these barcodes. Here, to address this, we developed an antigen-binding fragment (Fab7) that recognizes both active arrestin2 (β-arrestin1) and arrestin3 (β-arrestin2) without interacting with bound receptor polypeptides. We used Fab7 to determine the structures of both arrestins in complex with atypical chemokine receptor 3 (ACKR3) phosphorylated in different regions of its C-terminal tail by either GRK2 or GRK5 (ref. ). The GRK2-phosphorylated ACKR3 resulted in more heterogeneous 'tail-mode' assemblies, whereas phosphorylation by GRK5 resulted in more rigid 'ACKR3-adjacent' assemblies. Unexpectedly, the finger loops of both arrestins engaged the micelle surface rather than the receptor intracellular pocket, with arrestin3 being more dynamic, partly because of its lack of a membrane-anchoring motif. Thus, both the region of the barcode and the arrestin isoform involved can alter the structure and dynamics of GPCR-arrestin complexes, providing a possible mechanistic basis for unique downstream cellular effects, such as the efficiency of chemokine scavenging and the robustness of arrestin binding in ACKR3.
History
DepositionJul 19, 2023-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41289.png

Downloads & links

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Map

FileDownload / File: emd_41289.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.592
Minimum - Maximum-6.1378865 - 7.0237026
Average (Standard dev.)0.0006499817 (±0.102517314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_41289_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41289_half_map_2.map
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Sample components

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Entire : human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in n...

EntireName: human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in nanodisc
Components
  • Complex: human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in nanodisc
    • Protein or peptide: Beta-arrestin-1
    • Protein or peptide: Fab7 heavy chain
    • Protein or peptide: Fab7 light chain
    • Protein or peptide: anti-Fab nanobody
    • Protein or peptide: Atypical chemokine receptor 3

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Supramolecule #1: human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in n...

SupramoleculeName: human ACKR3 phosphorylated by GRK2 in complex with Arrestin2 in nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (domestic cattle)

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Macromolecule #1: Beta-arrestin-1

MacromoleculeName: Beta-arrestin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 47.055469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVEPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI ...String:
MGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVEPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI RKVQYAPERP GPQPTAETTR QFLMSDKPLH LEASLDKEIY YHGEPISVNV HVTNNTNKTV KKIKISVRQY AD ICLFNTA QYKCPVAMEE ADDTVAPSST FCKVYTLTPF LANNREKRGL ALDGKLKHED TNLASSTLLR EGANREILGI IVS YKVKVK LVVSRGGLLG DLASSDVAVE LPFTLMHPKP KEEPPHREVP EHETPVDTNL IELDTNDDDA AAEDFARQRL KGMK DDKEE EEDGTGSPRL NDR

UniProtKB: Beta-arrestin-1

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Macromolecule #2: Fab7 heavy chain

MacromoleculeName: Fab7 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 25.720758 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSSSYIHWV RQAPGKGLEW VASISSYYGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARKSMYHRGW GWLSWVYGAM DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVSSSYIHWV RQAPGKGLEW VASISSYYGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARKSMYHRGW GWLSWVYGAM DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC L VKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HT

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Macromolecule #3: Fab7 light chain

MacromoleculeName: Fab7 light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.471031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYYYPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYYYPITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: anti-Fab nanobody

MacromoleculeName: anti-Fab nanobody / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.390644 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSQVQLQESG GGLVQPGGSL RLSCAASGRT ISRYAMSWFR QAPGKEREFV AVARRSGDGA FYADSVQGRF TVSRDDAKNT VYLQMNSLK PEDTAVYYCA IDSDTFYSGS YDYWGQGTQV TVSS

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Macromolecule #5: Atypical chemokine receptor 3

MacromoleculeName: Atypical chemokine receptor 3 / type: protein_or_peptide / ID: 5 / Details: phosphorylated by GRK2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.356363 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GAPDLHLFDY SEPGNFSDIS WPCNSSDCIV VDTVMCPNMP NKSVLLYTLS FIYIFIFVIG MIANSVVVWV NIQAKTTGYD THCYILNLA IADLWVVLTI PVWVVSLVQH NQWPMGELTC KVTHLIFSIN LFGSIFFLTC MSVDRYLSIT YFTNTPSSRK K MVRRVVCI ...String:
GAPDLHLFDY SEPGNFSDIS WPCNSSDCIV VDTVMCPNMP NKSVLLYTLS FIYIFIFVIG MIANSVVVWV NIQAKTTGYD THCYILNLA IADLWVVLTI PVWVVSLVQH NQWPMGELTC KVTHLIFSIN LFGSIFFLTC MSVDRYLSIT YFTNTPSSRK K MVRRVVCI LVWLLAFCVS LPDTYYLKTV TSASNNETYC RSFYPEHSIK EWLIGMELVS VVLGFAVPFS IIAVFYFLLA RA ISASSDQ EKHSSRKIIF SYVVVFLVCW LPYHVAVLLD IFSILHYIPF TCRLEHALFT ALHVTQCLSL VHCCVNPVLY SFI NRNYRY ELMKAFIFKY SAKTGLTKLI DASRVSE(TPO)EY (SEP)ALEQSTKGR PLEVLFQGPH HHHHHHHHHD YKDDDD K

UniProtKB: Atypical chemokine receptor 3

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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

Concentration0.7 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 698683
Initial angle assignmentType: COMMON LINE / Software - Name: cryoSPARC
Final angle assignmentType: COMMON LINE / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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