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- PDB-9jfu: Cryo-EM structure of inactive GPR4 with NE52-QQ57 -

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Basic information

Entry
Database: PDB / ID: 9jfu
TitleCryo-EM structure of inactive GPR4 with NE52-QQ57
Components
  • Anti-bril fab light chain
  • Anti-fab nanobody
  • G-protein coupled receptor 4,Soluble cytochrome b562
  • Mbp-Anti-bril fab heavy chain
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / GPCR / Gs / Gsq / Proton sensing / SIGNALING PROTEIN / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


glomerular mesangial cell development / regulation of vascular permeability / Class A/1 (Rhodopsin-like receptors) / response to acidic pH / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / electron transport chain / G protein-coupled receptor activity ...glomerular mesangial cell development / regulation of vascular permeability / Class A/1 (Rhodopsin-like receptors) / response to acidic pH / angiogenesis involved in wound healing / positive regulation of Rho protein signal transduction / regulation of cell adhesion / negative regulation of angiogenesis / electron transport chain / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of inflammatory response / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (q) signalling events / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / iron ion binding / heme binding / plasma membrane
Similarity search - Function
G protein-coupled receptor 4 orphan / 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 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsYue, X.L. / Wu, L.J. / Hua, T. / Liu, Z.J.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2025
Title: Structural basis of stepwise proton sensing-mediated GPCR activation.
Authors: Xiaolei Yue / Li Peng / Shenhui Liu / Bingjie Zhang / Xiaodan Zhang / Hao Chang / Yuan Pei / Xiaoting Li / Junlin Liu / Wenqing Shui / Lijie Wu / Huji Xu / Zhi-Jie Liu / Tian Hua /
Abstract: The regulation of pH homeostasis is crucial in many biological processes vital for survival, growth, and function of life. The pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 ...The regulation of pH homeostasis is crucial in many biological processes vital for survival, growth, and function of life. The pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 and GPR68, play a pivotal role in detecting changes in extracellular proton concentrations, impacting both physiological and pathological states. However, comprehensive understanding of the proton sensing mechanism is still elusive. Here, we determined the cryo-electron microscopy structures of GPR4 and GPR65 in various activation states across different pH levels, coupled with G, G or G proteins, as well as a small molecule NE52-QQ57-bound inactive GPR4 structure. These structures reveal the dynamic nature of the extracellular loop 2 and its signature conformations in different receptor states, and disclose the proton sensing mechanism mediated by networks of extracellular histidine and carboxylic acid residues. Notably, we unexpectedly captured partially active intermediate states of both GPR4-G and GPR4-G complexes, and identified a unique allosteric binding site for NE52-QQ57 in GPR4. By integrating prior investigations with our structural analysis and mutagenesis data, we propose a detailed atomic model for stepwise proton sensation and GPCR activation. These insights may pave the way for the development of selective ligands and targeted therapeutic interventions for pH sensing-relevant diseases.
History
DepositionSep 5, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Mbp-Anti-bril fab heavy chain
K: Anti-fab nanobody
L: Anti-bril fab light chain
R: G-protein coupled receptor 4,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,0225
Polymers158,6064
Non-polymers4171
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Mbp-Anti-bril fab heavy chain


Mass: 66624.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#2: Antibody Anti-fab nanobody


Mass: 13390.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Anti-bril fab light chain


Mass: 23353.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#4: Protein G-protein coupled receptor 4,Soluble cytochrome b562 / G-protein coupled receptor 6C.l / GPR6C.l / Cytochrome b-562


Mass: 55236.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GPR4, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P46093, UniProt: P0ABE7
#5: Chemical ChemComp-A1L1E / NE52-QQ57 / 2-[4-[(2-ethyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl)methyl]phenyl]-5-piperidin-4-yl-1,3,4-oxadiazole


Mass: 416.519 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H28N6O / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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
1GPR4_antagonistCOMPLEX#1-#40RECOMBINANT
2fabCOMPLEX#1-#31RECOMBINANT
3GPR4-ICL3 BRILCOMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
33Escherichia coli (E. coli)562
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151171 / Symmetry type: POINT
RefinementHighest resolution: 3.23 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047486
ELECTRON MICROSCOPYf_angle_d0.64210177
ELECTRON MICROSCOPYf_dihedral_angle_d4.6661033
ELECTRON MICROSCOPYf_chiral_restr0.0421123
ELECTRON MICROSCOPYf_plane_restr0.0051290

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