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- PDB-9ik1: Cryo-EM structure of the human P2X3 receptor-compound 26a complex -

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Basic information

Entry
Database: PDB / ID: 9ik1
TitleCryo-EM structure of the human P2X3 receptor-compound 26a complex
ComponentsP2X purinoceptor 3
KeywordsMEMBRANE PROTEIN / P2X3 receptor / compound 26a / Cryo-EM
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 / cellular response to ATP ...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 / cellular response to ATP / positive regulation of calcium ion transport into cytosol / protein homotrimerization / behavioral response to pain / response to mechanical stimulus / positive regulation of calcium-mediated signaling / response to cold / 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
: / ADENOSINE-5'-TRIPHOSPHATE / P2X purinoceptor 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsKim, S. / Kim, G.R. / Kim, Y.O. / Han, X. / Nagel, J. / Kim, J. / Song, D.I. / Muller, C.E. / Yoon, M.H. / Jin, M.S. / Kim, Y.C.
Funding support Korea, Republic Of, 1items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 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

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Assembly

Deposited unit
A: P2X purinoceptor 3
B: P2X purinoceptor 3
C: P2X purinoceptor 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,07621
Polymers121,1213
Non-polymers4,95518
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein P2X purinoceptor 3 / P2X3 / ATP receptor / Purinergic receptor


Mass: 40373.520 Da / Num. of mol.: 3 / Mutation: T13P/S15V/V16I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: P2RX3 / Cell line (production host): HEK293S-GnTi / Production host: Homo sapiens (human) / References: UniProt: P56373
#2: Chemical ChemComp-A1L2M / 4-[2-cyclopropyl-7-[[(1~{R})-1-naphthalen-2-ylethyl]amino]-[1,2,4]triazolo[1,5-a]pyrimidin-5-yl]piperazine-1-carboxamide


Mass: 456.543 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C25H28N8O / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: 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.: 9 / Source method: obtained synthetically / 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
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / 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: Human P2X3 receptor-compound 26a complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
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: 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: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 588130 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0068473
ELECTRON MICROSCOPYf_angle_d0.80411528
ELECTRON MICROSCOPYf_dihedral_angle_d22.1091303
ELECTRON MICROSCOPYf_chiral_restr0.0521319
ELECTRON MICROSCOPYf_plane_restr0.0061420

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