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- EMDB-34983: Orphan GPR20 in complex with Fab046 -

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Basic information

Entry
Database: EMDB / ID: EMD-34983
TitleOrphan GPR20 in complex with Fab046
Map data
Sample
  • Complex: The complex of GPR20 with Fab046
    • Protein or peptide: heavy chain of Fab046
    • Protein or peptide: Light chain of Fab046
    • Protein or peptide: Soluble cytochrome b562,G-protein coupled receptor 20
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 speciesHomo sapiens (human) / Rattus (rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsLin X / Jiang S / Xu F
Funding support1 items
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, 2022-
Header (metadata) releaseMar 8, 2023-
Map releaseMar 8, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34983.png

Downloads & links

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Map

FileDownload / File: emd_34983.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.078
Minimum - Maximum-0.24956585 - 0.4854521
Average (Standard dev.)0.00016945104 (±0.012470375)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.616 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34983_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34983_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34983_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 : The complex of GPR20 with Fab046

EntireName: The complex of GPR20 with Fab046
Components
  • Complex: The complex of GPR20 with Fab046
    • Protein or peptide: heavy chain of Fab046
    • Protein or peptide: Light chain of Fab046
    • Protein or peptide: Soluble cytochrome b562,G-protein coupled receptor 20

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Supramolecule #1: The complex of GPR20 with Fab046

SupramoleculeName: The complex of GPR20 with Fab046 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: heavy chain of Fab046

MacromoleculeName: heavy chain of Fab046 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus (rat)
Molecular weightTheoretical: 26.493723 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEWNWVFLFL LSVTAEVHSQ VQLQQSGAEL AKPGSSVKIS CKASGYTFTS YYISWIKQTT GQGLKYIGFI NPGSGHTNYN EKFKGKATL TVDKSSSTAF MQLSSLTPDD SAIYYCARGA GGFLRIITKF DYWGQGVMVT VSSAQTTAPS VYPLAPGCGD T TSSTVTLG ...String:
MEWNWVFLFL LSVTAEVHSQ VQLQQSGAEL AKPGSSVKIS CKASGYTFTS YYISWIKQTT GQGLKYIGFI NPGSGHTNYN EKFKGKATL TVDKSSSTAF MQLSSLTPDD SAIYYCARGA GGFLRIITKF DYWGQGVMVT VSSAQTTAPS VYPLAPGCGD T TSSTVTLG CLVKGYFPEP VTVTWNSGAL SSDVHTFPAV LQSGLYTLTS SVTSSTWPSQ TVTCNVAHPA SSTKVDKKVG GS GHHHHHH

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Macromolecule #2: Light chain of Fab046

MacromoleculeName: Light chain of Fab046 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus (rat)
Molecular weightTheoretical: 25.637654 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: METDRLLLWV LLLWVPGSTG DTVLTQSPAL AVSLGQRVTI SCRASKSVST YIHWYQQRSG QQPKLLIYSA SNLESGVPSR FSGSGSGTD FTLTIDPVEP DDIANYYCQQ INELPYTFGA GTKLELKRAD AAPTVSIFPP STERLATGGA SVVCLMNNFY P RDISVKWK ...String:
METDRLLLWV LLLWVPGSTG DTVLTQSPAL AVSLGQRVTI SCRASKSVST YIHWYQQRSG QQPKLLIYSA SNLESGVPSR FSGSGSGTD FTLTIDPVEP DDIANYYCQQ INELPYTFGA GTKLELKRAD AAPTVSIFPP STERLATGGA SVVCLMNNFY P RDISVKWK IDGTERRDGV LDSVTDQDSK DSTYSMSSTL SLTKADYESH NLYTCEVVHK TSSSPVVKSF NRNEC

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Macromolecule #3: Soluble cytochrome b562,G-protein coupled receptor 20

MacromoleculeName: Soluble cytochrome b562,G-protein coupled receptor 20 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.631012 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAGRAADL EDNWETLNDN LKVIEKADNA AQVKDALTKM RAAALDAQKA TPPKLEDKSP DSPEMKDFR HGFDILVGQI DDALKLANEG KVKEAQAAAE QLKTTRNAYI QKYLMPSVSP AGPSAGAVPN ATAVTTVRTN A SGLEVPLF ...String:
MKTIIALSYI FCLVFADYKD DDDAGRAADL EDNWETLNDN LKVIEKADNA AQVKDALTKM RAAALDAQKA TPPKLEDKSP DSPEMKDFR HGFDILVGQI DDALKLANEG KVKEAQAAAE QLKTTRNAYI QKYLMPSVSP AGPSAGAVPN ATAVTTVRTN A SGLEVPLF HLFARLDEEL HGTFPGLWLA LMAVHGAIFL AGLVLNGLAL YVFCCRTRAK TPSVIYTINL VVTDLLVGLS LP TRFAVYY GARGCLRCAF PHVLGYFLNM HCSIWFLTCI CVDRYLAIVR PEGSRRCRQP ACARAVCAFV WLAAGAVTLS VLG VTGSRP CCRVFALTVL EFLLPLLVIS VFTGRIMCAL SRPGLLHQGR QRRVRAMQLL LTVLIIFLVC FTPFHARQVA VALW PDMPH HTSLVVYHVA VTLSSLNSCM NPIVYCFVTS GFQATVRGLF GQHGHHHHHH HHHH

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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: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 418288

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