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- EMDB-26135: Cryo-EM structure of GSK682753A-bound EBI2/GPR183 -

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Basic information

Entry
Database: EMDB / ID: EMD-26135
TitleCryo-EM structure of GSK682753A-bound EBI2/GPR183
Map dataGSK682753A-bound EBI2/GPR183
Sample
  • Complex: complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BRIL Fab and anti-Fab nanobody
    • Complex: anti-BRIL Fab Heavy chain, anti-BRIL Fab Light chain
      • Protein or peptide: anti-BRIL Fab Heavy chain
      • Protein or peptide: anti-BRIL Fab Light chain
    • Complex: anti-Fab Nanobody
      • Protein or peptide: anti-Fab Nanobody
    • Complex: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
      • Protein or peptide: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
  • Ligand: 8-[(2E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)methyl]-1-oxa-3,8-diazaspiro[4.5]decan-2-one
Function / homology
Function and homology information


regulation of astrocyte chemotaxis / B cell activation involved in immune response / dendritic cell homeostasis / T follicular helper cell differentiation / mature B cell differentiation involved in immune response / Class A/1 (Rhodopsin-like receptors) / T cell chemotaxis / oxysterol binding / leukocyte chemotaxis / dendritic cell chemotaxis ...regulation of astrocyte chemotaxis / B cell activation involved in immune response / dendritic cell homeostasis / T follicular helper cell differentiation / mature B cell differentiation involved in immune response / Class A/1 (Rhodopsin-like receptors) / T cell chemotaxis / oxysterol binding / leukocyte chemotaxis / dendritic cell chemotaxis / plasma membrane => GO:0005886 / humoral immune response / positive regulation of B cell proliferation / osteoclast differentiation / G protein-coupled receptor activity / G alpha (i) signalling events / adaptive immune response / positive regulation of ERK1 and ERK2 cascade / immune response / G protein-coupled receptor signaling pathway / plasma membrane
Similarity search - Function
Serpentine type 7TM GPCR chemoreceptor Srsx / 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
G-protein coupled receptor 183
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsChen H / Huang W / Li X
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01HL020948 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM135343 United States
Damon Runyon Cancer Research FoundationDRR-53S-19 United States
CitationJournal: Structure / Year: 2022
Title: Structures of oxysterol sensor EBI2/GPR183, a key regulator of the immune response.
Authors: Hongwen Chen / Weijiao Huang / Xiaochun Li /
Abstract: Oxysterols induce the migration of B-lymphocytes and dendritic cells to interfollicular regions of lymphoid tissues through binding the EBI2 (GPR183) to stimulate effective adaptive immunity and ...Oxysterols induce the migration of B-lymphocytes and dendritic cells to interfollicular regions of lymphoid tissues through binding the EBI2 (GPR183) to stimulate effective adaptive immunity and antibody production during infection. Aberrant EBI2 signaling is implicated in inflammatory bowel disease, sclerosis, and infectious disease. Here, we report the cryo-EM structures of an EBI2-G signaling complex with its endogenous agonist 7α,25-OHC and that of an inactive EBI2 bound to the inverse agonist GSK682753A. These structures reveal an agonist binding site for the oxysterol and a potential ligand entrance site exposed to the lipid bilayer. Mutations within the oxysterol binding site and the Gα interface attenuate G protein signaling and abolish oxysterol-mediated cell migration indicating that G protein signaling directly involves in the oxysterol-EBI2 pathway. Together, these findings provide new insight into how EBI2 is activated by an oxysterol ligand and will facilitate the development of therapeutic approaches that target EBI2-linked diseases.
History
DepositionFeb 3, 2022-
Header (metadata) releaseApr 13, 2022-
Map releaseApr 13, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26135.png

Downloads & links

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Map

FileDownload / File: emd_26135.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGSK682753A-bound EBI2/GPR183
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.38
Minimum - Maximum-1.2770617 - 2.6706774
Average (Standard dev.)0.005848937 (±0.054565415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BR...

EntireName: complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BRIL Fab and anti-Fab nanobody
Components
  • Complex: complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BRIL Fab and anti-Fab nanobody
    • Complex: anti-BRIL Fab Heavy chain, anti-BRIL Fab Light chain
      • Protein or peptide: anti-BRIL Fab Heavy chain
      • Protein or peptide: anti-BRIL Fab Light chain
    • Complex: anti-Fab Nanobody
      • Protein or peptide: anti-Fab Nanobody
    • Complex: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
      • Protein or peptide: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
  • Ligand: 8-[(2E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)methyl]-1-oxa-3,8-diazaspiro[4.5]decan-2-one

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Supramolecule #1: complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BR...

SupramoleculeName: complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BRIL Fab and anti-Fab nanobody
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: anti-BRIL Fab Heavy chain, anti-BRIL Fab Light chain

SupramoleculeName: anti-BRIL Fab Heavy chain, anti-BRIL Fab Light chain / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1, #3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #3: anti-Fab Nanobody

SupramoleculeName: anti-Fab Nanobody / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Supramolecule #4: G-protein coupled receptor 183,Soluble cytochrome b562 fusion

SupramoleculeName: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
type: complex / Chimera: Yes / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.321084 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVVDFSLHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK S

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Macromolecule #2: anti-Fab Nanobody

MacromoleculeName: anti-Fab Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.071431 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
HHHHHHGENL YFQGSQVQLQ ESGGGLVQPG GSLRLSCAAS GRTISRYAMS WFRQAPGKER EFVAVARRSG DGAFYADSVQ GRFTVSRDD AKNTVYLQMN SLKPEDTAVY YCAIDSDTFY SGSYDYWGQG TQVTVSS

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Macromolecule #3: anti-BRIL Fab Light chain

MacromoleculeName: anti-BRIL Fab Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.483062 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGE

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Macromolecule #4: G-protein coupled receptor 183,Soluble cytochrome b562 fusion

MacromoleculeName: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.364539 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDIQMANNFT PPSATPQGND CDLYAHHSTA RIVMPLHYSL VFIIGLVGNL LALVVIVQNR KKINSTTLYS TNLVISDILF TTALPTRIA YYAMGFDWRI GDALCRITAL VFYINTYAGV NFMTCLSIDR FIAVVHPLRY NKIKRIEHAK GVCIFVWILV F AQTLPLLI ...String:
MDIQMANNFT PPSATPQGND CDLYAHHSTA RIVMPLHYSL VFIIGLVGNL LALVVIVQNR KKINSTTLYS TNLVISDILF TTALPTRIA YYAMGFDWRI GDALCRITAL VFYINTYAGV NFMTCLSIDR FIAVVHPLRY NKIKRIEHAK GVCIFVWILV F AQTLPLLI NPMSKQEAER ITCMEYPNFE ETKSLPWILL GACFIGYVLP LIIILICYSQ ICCKLFRARR QLADLEDNWE TL NDNLKVI EKADNAAQVK DALTKMRAAA LDAQKATPPK LEDKSPDSPE MKDFRHGFDI LVGQIDDALK LANEGKVKEA QAA AEQLKT TRNAYIQKYL ERARSTLSGV NKKALNTIIL IIVVFVLCFT PYHVAIIQHM IKKLRFSNFL ECSQRHSFQI SLHF TVCLM NFNCCMDPFI YFFACKGYKR KVMRMLKRQV SVSISSAVKS APEENSREMT ETQMMIHSKS SNGKDYKDDD DK

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Macromolecule #5: 8-[(2E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)me...

MacromoleculeName: 8-[(2E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)methyl]-1-oxa-3,8-diazaspiro[4.5]decan-2-one
type: ligand / ID: 5 / Number of copies: 1 / Formula: KKF
Molecular weightTheoretical: 479.783 Da
Chemical component information

ChemComp-KKF:
8-[(2E)-3-(4-chlorophenyl)prop-2-enoyl]-3-[(3,4-dichlorophenyl)methyl]-1-oxa-3,8-diazaspiro[4.5]decan-2-one

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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
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

21927

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 222283

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