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- PDB-9bpc: Cryo-EM structure of P2X3 receptor in complex with camlipixant -

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Basic information

Entry
Database: PDB / ID: 9bpc
TitleCryo-EM structure of P2X3 receptor in complex with camlipixant
ComponentsP2X purinoceptor
KeywordsMEMBRANE PROTEIN / P2X3 / camlipixant / cryo-EM / ion channel / receptor
Function / homology
Function and homology information


extracellularly ATP-gated monoatomic cation channel activity / purinergic nucleotide receptor activity / response to ATP / postsynapse / 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
: / P2X purinoceptor
Similarity search - Component
Biological speciesCanis lupus (wolf)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsThach, T. / Subramanian, R.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2025
Title: Mechanistic insights into the selective targeting of P2X3 receptor by camlipixant antagonist.
Authors: Trung Thach / KanagaVijayan Dhanabalan / Prajwal Prabhakarrao Nandekar / Seth Stauffer / Iring Heisler / Sarah Alvarado / Jonathan Snyder / Ramaswamy Subramanian /
Abstract: ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts ...ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts to develop selective drugs targeting P2X3 have led to the development of camlipixant, a potent, selective P2X3 antagonist. However, the mechanisms of receptor desensitization, ion permeation, and structural basis of camlipixant binding to P2X3 remain unclear. Here, we report a cryo-EM structure of camlipixant-bound P2X3, revealing a previously undiscovered selective drug-binding site in the receptor. Our findings also demonstrate that conformational changes in the upper body domain, including the turret and camlipixant-binding pocket, play a critical role: turret opening facilitates P2X3 channel closure to a radius of 0.7 Å, hindering cation transfer, whereas turret closure leads to channel opening. Structural and functional studies combined with molecular dynamics simulations provide a comprehensive understanding of camlipixant's selective inhibition of P2X3, offering a foundation for future drug development targeting this receptor.
History
DepositionMay 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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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)124,74512
Polymers121,4333
Non-polymers3,3129
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


Mass: 40477.520 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Canis lupus (wolf) / Gene: P2RX3 / Production host: Homo sapiens (human) / References: UniProt: A0A8I3S575
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Chemical ChemComp-A1AQX / Camlipixant


Mass: 458.458 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C23H24F2N4O4 / Feature type: SUBJECT OF INVESTIGATION
#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.: 3 / 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
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: Ternary complex of P2X3 receptor and camlipixant / Type: COMPLEX / Details: P2X3 receptor and camlipixant antagonist / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.126 MDa / Experimental value: YES
Source (natural)Organism: Canis lupus (wolf)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 20 mM HEPES, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2100 mMSodium ChlorideNaCl1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: P2X3 was incubated with camlipixant in a molar ratio of 1:5 for 30 minutes on ice before being used to prepare cryo-EM grids
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: vitrification

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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: 700 nm / Cs: 0.1 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 56.8 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 60
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
7PHENIX1.12.1model fitting
12cryoSPARC4.4.13D reconstruction
13PHENIX1.21.1_5286:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 200982
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40142 / Num. of class averages: 50 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Details: real refinement was done using Phenix
Atomic model buildingChain residue range: 1-361 / Details: The initial model consisted of monomer / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027539
ELECTRON MICROSCOPYf_angle_d0.54810242
ELECTRON MICROSCOPYf_dihedral_angle_d7.5941248
ELECTRON MICROSCOPYf_chiral_restr0.0461158
ELECTRON MICROSCOPYf_plane_restr0.0031299

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