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- PDB-8jv7: Cryo-EM structure of the panda P2X7 receptor in complex with PPADS -

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Basic information

Entry
Database: PDB / ID: 8jv7
TitleCryo-EM structure of the panda P2X7 receptor in complex with PPADS
ComponentsP2X purinoceptor
KeywordsTRANSPORT PROTEIN / Channel
Function / homology
Function and homology information


purinergic nucleotide receptor activity / extracellularly ATP-gated monoatomic cation channel activity / response to ATP / postsynapse / ATP binding / plasma membrane
Similarity search - Function
P2X purinoreceptor 7, intracellular domain / P2X purinoreceptor 7 intracellular domain / P2X7 purinoceptor / ATP P2X receptor / ATP P2X receptors signature. / P2X purinoreceptor / P2X purinoreceptor extracellular domain superfamily
Similarity search - Domain/homology
Chem-UO6 / P2X purinoceptor
Similarity search - Component
Biological speciesAiluropoda melanoleuca (giant panda)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSheng, D. / Hattori, M.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
Citation
Journal: Elife / Year: 2024
Title: Structural insights into the orthosteric inhibition of P2X receptors by non-ATP analog antagonists.
Authors: Danqi Sheng / Chen-Xi Yue / Fei Jin / Yao Wang / Muneyoshi Ichikawa / Ye Yu / Chang-Run Guo / Motoyuki Hattori /
Abstract: P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, ...P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.
#1: Journal: Elife / Year: 2023
Title: Structural insights into the orthosteric inhibition of P2X receptors by non-ATP-analog antagonists
Authors: Sheng, D. / Yue, C. / Jin, F. / Wang, Y. / Ichikawa, M. / Yu, Y. / Guo, C.R. / Hattori, M.
History
DepositionJun 27, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2May 15, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)119,01012
Polymers116,1493
Non-polymers2,8619
Water543
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: 38716.234 Da / Num. of mol.: 3 / Mutation: V35A, R125A, E174K, N241S, N284S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ailuropoda melanoleuca (giant panda) / Gene: PANDA_000894 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: D2GVW0
#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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
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-UO6 / 4-[(E)-[4-methanoyl-6-methyl-5-oxidanyl-3-(phosphonooxymethyl)pyridin-2-yl]diazenyl]benzene-1,3-disulfonic acid / Ppads


Mass: 511.378 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C14H14N3O12PS2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: P2X7 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Ailuropoda melanoleuca (giant panda)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 50 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: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161188 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037434
ELECTRON MICROSCOPYf_angle_d0.52210158
ELECTRON MICROSCOPYf_dihedral_angle_d4.9721080
ELECTRON MICROSCOPYf_chiral_restr0.0441125
ELECTRON MICROSCOPYf_plane_restr0.0051302

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