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- PDB-8jv6: Cryo-EM structure of the zebrafish P2X4 receptor in complex with ... -

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Basic information

Entry
Database: PDB / ID: 8jv6
TitleCryo-EM structure of the zebrafish P2X4 receptor in complex with BAY-1797
ComponentsP2X purinoceptor
KeywordsTRANSPORT PROTEIN / ATP / allosteric modulator / channel
Function / homology
Function and homology information


Elevation of cytosolic Ca2+ levels / Platelet homeostasis / purine nucleotide binding / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / ATP-gated ion channel activity / CTP binding / monoatomic ion channel complex / response to ATP / monoatomic cation transport ...Elevation of cytosolic Ca2+ levels / Platelet homeostasis / purine nucleotide binding / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / ATP-gated ion channel activity / CTP binding / monoatomic ion channel complex / response to ATP / monoatomic cation transport / ligand-gated monoatomic ion channel activity / transmembrane transporter complex / calcium ion transmembrane transport / calcium ion transport / postsynapse / lysosomal membrane / ATP binding / membrane / plasma membrane
Similarity search - Function
P2X4 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
Chem-P6E / P2X purinoceptor 4a
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsHattori, M. / Shen, C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2023
Title: Structural insights into the allosteric inhibition of P2X4 receptors.
Authors: Cheng Shen / Yuqing Zhang / Wenwen Cui / Yimeng Zhao / Danqi Sheng / Xinyu Teng / Miaoqing Shao / Muneyoshi Ichikawa / Jin Wang / Motoyuki Hattori /
Abstract: P2X receptors are ATP-activated cation channels, and the P2X4 subtype plays important roles in the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 ...P2X receptors are ATP-activated cation channels, and the P2X4 subtype plays important roles in the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 receptors are of increasing interest as potential drug targets. Here, we report the cryo-EM structures of the zebrafish P2X4 receptor in complex with two P2X4 subtype-specific antagonists, BX430 and BAY-1797. Both antagonists bind to the same allosteric site located at the subunit interface at the top of the extracellular domain. Structure-based mutational analysis by electrophysiology identified the important residues for the allosteric inhibition of both zebrafish and human P2X4 receptors. Structural comparison revealed the ligand-dependent structural rearrangement of the binding pocket to stabilize the binding of allosteric modulators, which in turn would prevent the structural changes of the extracellular domain associated with channel activation. Furthermore, comparison with the previously reported P2X structures of other subtypes provided mechanistic insights into subtype-specific allosteric inhibition.
History
DepositionJun 27, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 24, 2024Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: P2X purinoceptor
B: P2X purinoceptor
C: P2X purinoceptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,99718
Polymers118,0913
Non-polymers3,90515
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: Protein P2X purinoceptor


Mass: 39363.832 Da / Num. of mol.: 3 / Mutation: H252R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: p2rx4a / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6NYR1
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-P6E / N-[4-(3-chloranylphenoxy)-3-sulfamoyl-phenyl]-2-phenyl-ethanamide


Mass: 416.878 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C20H17ClN2O4S / 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

ComponentName: P2X4 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
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: 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: 46 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 205814 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037623
ELECTRON MICROSCOPYf_angle_d0.53210398
ELECTRON MICROSCOPYf_dihedral_angle_d7.0051113
ELECTRON MICROSCOPYf_chiral_restr0.0441185
ELECTRON MICROSCOPYf_plane_restr0.0051350

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