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- PDB-8hs2: Orphan GPR20 in complex with Fab046 -

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Basic information

Entry
Database: PDB / ID: 8hs2
TitleOrphan GPR20 in complex with Fab046
Components
  • Light chain of Fab046
  • Soluble cytochrome b562,G-protein coupled receptor 20
  • heavy chain of Fab046
KeywordsMEMBRANE PROTEIN / Complex / GPR20 / GPCR / orphan / orphan receptor / class-A
Function / homology
Function and homology information


positive regulation of Rho protein signal transduction / G protein-coupled receptor activity / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (s) signalling events / electron transfer activity / periplasmic space / receptor complex / iron ion binding / heme binding / plasma membrane / cytosol
Similarity search - Function
Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
Soluble cytochrome b562 / G-protein coupled receptor 20
Similarity search - Component
Biological speciesRattus (rat)
Escherichia coli (E. coli)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLin, X. / Jiang, S. / Xu, F.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Discov / Year: 2023
Title: The activation mechanism and antibody binding mode for orphan GPR20.
Authors: Xi Lin / Shan Jiang / Yiran Wu / Xiaohu Wei / Gye-Won Han / Lijie Wu / Junlin Liu / Bo Chen / Zhibin Zhang / Suwen Zhao / Vadim Cherezov / Fei Xu /
Abstract: GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug ...GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug conjugate (ADC) containing a GPR20-binding antibody (Ab046) was recently developed in clinical trials for GIST treatment. GPR20 constitutively activates Gi proteins in the absence of any known ligand, but it remains obscure how this high basal activity is achieved. Here we report three cryo-EM structures of human GPR20 complexes including Gi-coupled GPR20 in the absence or presence of the Fab fragment of Ab046 and Gi-free GPR20. Remarkably, the structures demonstrate a uniquely folded N-terminal helix capping onto the transmembrane domain and our mutagenesis study suggests a key role of this cap region in stimulating the basal activity of GPR20. We also uncover the molecular interactions between GPR20 and Ab046, which may enable the design of tool antibodies with enhanced affinity or new functionality for GPR20. Furthermore, we report the orthosteric pocket occupied by an unassigned density which might be essential for exploring opportunities for deorphanization.
History
DepositionDec 16, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: heavy chain of Fab046
C: Light chain of Fab046
R: Soluble cytochrome b562,G-protein coupled receptor 20


Theoretical massNumber of molelcules
Total (without water)102,7623
Polymers102,7623
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody heavy chain of Fab046


Mass: 26493.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus (rat) / Production host: Spodoptera frugiperda (fall armyworm)
#2: Antibody Light chain of Fab046


Mass: 25637.654 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus (rat) / Production host: Spodoptera frugiperda (fall armyworm)
#3: Protein Soluble cytochrome b562,G-protein coupled receptor 20 / Cytochrome b-562


Mass: 50631.012 Da / Num. of mol.: 1 / Mutation: L139W,D293N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, GPR20 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P0ABE7, UniProt: Q99678

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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

ComponentName: The complex of GPR20 with Fab046 / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
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: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 418288 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045587
ELECTRON MICROSCOPYf_angle_d0.7667613
ELECTRON MICROSCOPYf_dihedral_angle_d4.459774
ELECTRON MICROSCOPYf_chiral_restr0.046892
ELECTRON MICROSCOPYf_plane_restr0.005954

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