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- PDB-7tuy: Cryo-EM structure of GSK682753A-bound EBI2/GPR183 -

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Basic information

Entry
Database: PDB / ID: 7tuy
TitleCryo-EM structure of GSK682753A-bound EBI2/GPR183
Components
  • G-protein coupled receptor 183,Soluble cytochrome b562 fusion
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
  • anti-Fab Nanobody
KeywordsIMMUNE SYSTEM / EBI2 / GPR183 / oxysterol / GPCR / signaling / immunity / immunocyte migration
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
Chem-KKF / G-protein coupled receptor 183
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsChen, H. / Huang, W. / Li, X.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 13, 2022Provider: repository / Type: Initial release
Revision 1.1May 25, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: anti-BRIL Fab Heavy chain
K: anti-Fab Nanobody
L: anti-BRIL Fab Light chain
R: G-protein coupled receptor 183,Soluble cytochrome b562 fusion
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,7205
Polymers117,2404
Non-polymers4801
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody anti-BRIL Fab Heavy chain


Mass: 24321.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: Antibody anti-Fab Nanobody


Mass: 15071.431 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Antibody anti-BRIL Fab Light chain


Mass: 23483.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: Protein G-protein coupled receptor 183,Soluble cytochrome b562 fusion / Epstein-Barr virus-induced G-protein coupled receptor 2 / EBI2 / EBV-induced G-protein coupled ...Epstein-Barr virus-induced G-protein coupled receptor 2 / EBI2 / EBV-induced G-protein coupled receptor 2 / hEBI2


Mass: 54364.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: GPR183, EBI2 / Cell line (production host): HEK203S GnTI- / Production host: Homo sapiens (human) / References: UniProt: P32249
#5: Chemical 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


Mass: 479.783 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H21Cl3N2O3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
1complex of GSK682753A-bound EBI2/GPR183-BRIL chimera with anti-BRIL Fab and anti-Fab nanobodyCOMPLEX#1-#30RECOMBINANT
2anti-BRIL Fab Heavy chain, anti-BRIL Fab Light chainCOMPLEX#1, #31RECOMBINANT
3anti-Fab NanobodyCOMPLEX#21RECOMBINANT
4G-protein coupled receptor 183,Soluble cytochrome b562 fusionCOMPLEX#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
23Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
34Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: NONE
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 222283 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057315
ELECTRON MICROSCOPYf_angle_d0.8019939
ELECTRON MICROSCOPYf_dihedral_angle_d8.2874321
ELECTRON MICROSCOPYf_chiral_restr0.051135
ELECTRON MICROSCOPYf_plane_restr0.0061250

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