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- PDB-8tf3: Wildtype rabbit TRPV5 into nanodiscs in complex with econazole -

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Basic information

Entry
Database: PDB / ID: 8tf3
TitleWildtype rabbit TRPV5 into nanodiscs in complex with econazole
ComponentsTransient receptor potential cation channel subfamily V member 5
KeywordsMEMBRANE PROTEIN / TRPV5 / TRP channel / econazole
Function / homology
Function and homology information


regulation of urine volume / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel activity / calcium ion transmembrane transport / calcium ion transport / protein homotetramerization / calmodulin binding / apical plasma membrane / identical protein binding ...regulation of urine volume / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel activity / calcium ion transmembrane transport / calcium ion transport / protein homotetramerization / calmodulin binding / apical plasma membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 5 / Transient receptor potential cation channel subfamily V member 5/6 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily ...Transient receptor potential cation channel subfamily V member 5 / Transient receptor potential cation channel subfamily V member 5/6 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-CPL / Chem-ECN / ERGOSTEROL / Transient receptor potential cation channel subfamily V member 5
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsDe Jesus-Perez, J.J. / Fluck, E.C. / Moiseenkova-Bell, V.Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144120 United States
CitationJournal: Structure / Year: 2024
Title: Structural mechanism of TRPV5 inhibition by econazole.
Authors: José J De Jesús-Pérez / Matthew Gabrielle / Sumiyya Raheem / Edwin C Fluck / Tibor Rohacs / Vera Y Moiseenkova-Bell /
Abstract: The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular ...The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P, preventing the latter from binding, and ultimately pore closure.
History
DepositionJul 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 5
B: Transient receptor potential cation channel subfamily V member 5
C: Transient receptor potential cation channel subfamily V member 5
D: Transient receptor potential cation channel subfamily V member 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)345,90324
Polymers335,1384
Non-polymers10,76420
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 5 / TrpV5 / Epithelial calcium channel 1 / ECaC1 / Osm-9-like TRP channel 3 / OTRPC3


Mass: 83784.586 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: Trpv5, Ecac1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q9XSM3
#2: Chemical
ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C28H44O
#3: Chemical
ChemComp-CPL / 1-PALMITOYL-2-LINOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / PALMITOYL-LINOLEOYL PHOSPHATIDYLCHOLINE


Mass: 758.060 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C42H80NO8P / Comment: phospholipid*YM
#4: Chemical
ChemComp-ECN / 1-[(2S)-2-[(4-CHLOROBENZYL)OXY]-2-(2,4-DICHLOROPHENYL)ETHYL]-1H-IMIDAZOLE / S-Econazole


Mass: 381.684 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H15Cl3N2O / Feature type: SUBJECT OF INVESTIGATION
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: Tetramer of wildtype rabbit TRPV5 into nanodiscs in complex with econazole
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.33514 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6018

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.2particle selection
4cryoSPARC4.0.2CTF correction
7ISOLDEmodel fitting
8Coot0.9.8.3model fitting
10cryoSPARC4.0.2initial Euler assignment
11cryoSPARC4.0.2final Euler assignment
12cryoSPARC4.0.2classification
13cryoSPARC4.0.23D reconstruction
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1426748
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173323 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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