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- EMDB-36668: Cryo-EM structure of the zebrafish P2X4 receptor in complex with BX430 -

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Basic information

Entry
Database: EMDB / ID: EMD-36668
TitleCryo-EM structure of the zebrafish P2X4 receptor in complex with BX430
Map data
Sample
  • Complex: P2X4 trimer
    • Protein or peptide: P2X purinoceptor
  • Ligand: 1-[2,6-bis(bromanyl)-4-propan-2-yl-phenyl]-3-pyridin-3-yl-urea
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsATP / allosteric modulator / channel / TRANSPORT PROTEIN
Function / homology
Function and homology information


Elevation of cytosolic Ca2+ levels / Platelet homeostasis / purine nucleotide binding / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / ATP-gated ion channel activity / CTP binding / monoatomic ion channel complex / response to ATP / transmembrane transporter complex ...Elevation of cytosolic Ca2+ levels / Platelet homeostasis / purine nucleotide binding / extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / ATP-gated ion channel activity / CTP binding / monoatomic ion channel complex / response to ATP / transmembrane transporter complex / monoatomic cation transport / ligand-gated monoatomic ion channel activity / 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
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsHattori M / Shen C
Funding support China, 1 items
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, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36668.png

Downloads & links

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Map

FileDownload / File: emd_36668.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å		0.83 Å/pix.
x 256 pix.
= 212.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-3.7540026 - 4.3356314
Average (Standard dev.)-0.0011517252 (±0.094109654)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36668_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

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Half map: #2

Fileemd_36668_half_map_2.map
Projections & Slices
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Sample components

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Entire : P2X4 trimer

EntireName: P2X4 trimer
Components
  • Complex: P2X4 trimer
    • Protein or peptide: P2X purinoceptor
  • Ligand: 1-[2,6-bis(bromanyl)-4-propan-2-yl-phenyl]-3-pyridin-3-yl-urea
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: P2X4 trimer

SupramoleculeName: P2X4 trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: P2X purinoceptor

MacromoleculeName: P2X purinoceptor / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 39.476988 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DSVSQCFFDY YTSKILIIRS KKVGTLNRFT QALVIAYVIG YVCVYNKGYQ DTDTVLSSVT TKVKGIALTN TSELGERIWD VADYIIPPQ EDGSFFVLTN MIITTNQTQS KCAENPTPAS TCTSHRDCKR GFNDARGDGV RTGRCVSYSA SVKTCEVLSW C PLEKIVDP ...String:
DSVSQCFFDY YTSKILIIRS KKVGTLNRFT QALVIAYVIG YVCVYNKGYQ DTDTVLSSVT TKVKGIALTN TSELGERIWD VADYIIPPQ EDGSFFVLTN MIITTNQTQS KCAENPTPAS TCTSHRDCKR GFNDARGDGV RTGRCVSYSA SVKTCEVLSW C PLEKIVDP PNPPLLADAE NFTVLIKNNI RYPKFNFNKR NILPNINSSY LTHCVFSRKT DPDCPIFRLG DIVGEAEEDF QI MAVRGGV MGVQIRWDCD LDMPQSWCVP RYTFRRLDNK DPDNNVAPGY NFRFAKYYKN SDGTETRTLI KGYGIRFDVM VFG QAGKFN IIPTLLNIGA GLALLGLVNV ICDWI

UniProtKB: P2X purinoceptor 4a

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Macromolecule #2: 1-[2,6-bis(bromanyl)-4-propan-2-yl-phenyl]-3-pyridin-3-yl-urea

MacromoleculeName: 1-[2,6-bis(bromanyl)-4-propan-2-yl-phenyl]-3-pyridin-3-yl-urea
type: ligand / ID: 2 / Number of copies: 3 / Formula: P73
Molecular weightTheoretical: 413.107 Da
Chemical component information

ChemComp-P73:
1-[2,6-bis(bromanyl)-4-propan-2-yl-phenyl]-3-pyridin-3-yl-urea

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 9 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4dw0

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 372792
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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