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- EMDB-60647: Cryo-EM structure of the human P2X3 receptor-compound 26a complex -

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Basic information

Entry
Database: EMDB / ID: EMD-60647
TitleCryo-EM structure of the human P2X3 receptor-compound 26a complex
Map data
Sample
  • Complex: Human P2X3 receptor-compound 26a complex
    • Protein or peptide: P2X purinoceptor 3
  • Ligand: 4-[2-cyclopropyl-7-[[(1~{R})-1-naphthalen-2-ylethyl]amino]-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl]piperazine-1-carboxamide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
KeywordsP2X3 receptor / compound 26a / Cryo-EM / MEMBRANE PROTEIN
Function / homology
Function and homology information


Platelet homeostasis / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / neuromuscular synaptic transmission / Elevation of cytosolic Ca2+ levels / peristalsis / urinary bladder smooth muscle contraction / response to carbohydrate / inorganic cation transmembrane transport / positive regulation of calcium ion transport into cytosol ...Platelet homeostasis / purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / neuromuscular synaptic transmission / Elevation of cytosolic Ca2+ levels / peristalsis / urinary bladder smooth muscle contraction / response to carbohydrate / inorganic cation transmembrane transport / positive regulation of calcium ion transport into cytosol / cellular response to ATP / behavioral response to pain / protein homotrimerization / response to mechanical stimulus / response to cold / positive regulation of calcium-mediated signaling / hippocampal mossy fiber to CA3 synapse / establishment of localization in cell / calcium ion transmembrane transport / regulation of synaptic plasticity / Schaffer collateral - CA1 synapse / sensory perception of taste / response to heat / postsynapse / receptor complex / response to hypoxia / axon / signal transduction / ATP binding / plasma membrane
Similarity search - Function
P2X3 purinoceptor / : / ATP P2X receptors signature. / ATP P2X receptor / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsKim S / Kim GR / Kim YO / Han X / Nagel J / Kim J / Song DI / Muller CE / Yoon MH / Jin MS / Kim YC
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2022R1A2C1091278 Korea, Republic Of
CitationJournal: J Med Chem / Year: 2024
Title: Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy.
Authors: Ga-Ram Kim / Subin Kim / Yeo-Ok Kim / Xuehao Han / Jessica Nagel / Jihyun Kim / Dahin Irene Song / Christa E Müller / Myung-Ha Yoon / Mi Sun Jin / Yong-Chul Kim /
Abstract: The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., ...The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound was optimized through structure-activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist , featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that stabilizes the P2X3R in its desensitized state, acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent antiallodynic effects of , thus presenting a novel, promising lead structure.
History
DepositionJun 26, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60647.png

Downloads & links

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Map

FileDownload / File: emd_60647.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 250 pix.
= 242.5 Å		0.97 Å/pix.
x 250 pix.
= 242.5 Å		0.97 Å/pix.
x 250 pix.
= 242.5 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.97 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.078
Minimum - Maximum-0.29735062 - 0.74879724
Average (Standard dev.)0.0017253545 (±0.02073309)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 242.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_60647_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60647_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Human P2X3 receptor-compound 26a complex

EntireName: Human P2X3 receptor-compound 26a complex
Components
  • Complex: Human P2X3 receptor-compound 26a complex
    • Protein or peptide: P2X purinoceptor 3
  • Ligand: 4-[2-cyclopropyl-7-[[(1~{R})-1-naphthalen-2-ylethyl]amino]-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl]piperazine-1-carboxamide
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Human P2X3 receptor-compound 26a complex

SupramoleculeName: Human P2X3 receptor-compound 26a complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: P2X purinoceptor 3

MacromoleculeName: P2X purinoceptor 3 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.37352 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DFFTYETPKV IVVKSWTIGI INRVVQLLII SYFVGWVFLH EKAYQVRDTA IESSVVTKVK GSGLYANRVM DVSDYVTPPQ GTSVFVIIT KMIVTENQMQ GFCPESEEKY RCVSDSQCGP ERLPGGGILT GRCVNYSSVL RTCEIQGWCP TEVDTVETPI M MEAENFTI ...String:
DFFTYETPKV IVVKSWTIGI INRVVQLLII SYFVGWVFLH EKAYQVRDTA IESSVVTKVK GSGLYANRVM DVSDYVTPPQ GTSVFVIIT KMIVTENQMQ GFCPESEEKY RCVSDSQCGP ERLPGGGILT GRCVNYSSVL RTCEIQGWCP TEVDTVETPI M MEAENFTI FIKNSIRFPL FNFEKGNLLP NLTARDMKTC RFHPDKDPFC PILRVGDVVK FAGQDFAKLA RTGGVLGIKI GW VCDLDKA WDQCIPKYSF TRLDSVSEKS SVSPGYNFRF AKYYKMENGS EYRTLLKAFG IRFDVLVYGN AGKFNIIPTI ISS VAAFTS VGVGTVLCDI ILLNFLKGAD QYKAKKFEEV NET

UniProtKB: P2X purinoceptor 3

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Macromolecule #2: 4-[2-cyclopropyl-7-[[(1~{R})-1-naphthalen-2-ylethyl]amino]-[1,2,4...

MacromoleculeName: 4-[2-cyclopropyl-7-[[(1~{R})-1-naphthalen-2-ylethyl]amino]-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl]piperazine-1-carboxamide
type: ligand / ID: 2 / Number of copies: 3 / Formula: A1L2M
Molecular weightTheoretical: 456.543 Da

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / 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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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 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: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 588130
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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