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- PDB-7k4c: Cryo-EM structure of human TRPV6 in complex with (4- phenylcycloh... -

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Basic information

Entry
Database: PDB / ID: 7k4c
TitleCryo-EM structure of human TRPV6 in complex with (4- phenylcyclohexyl)piperazine inhibitor Br-cis-22a
ComponentsTransient receptor potential cation channel subfamily V member 6
KeywordsMEMBRANE PROTEIN/INHIBITOR / TRPV6 / ion channel / inhibitor / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


parathyroid hormone secretion / regulation of calcium ion-dependent exocytosis / TRP channels / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / response to calcium ion / calcium ion transmembrane transport / calcium channel activity / calcium ion transport ...parathyroid hormone secretion / regulation of calcium ion-dependent exocytosis / TRP channels / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / response to calcium ion / calcium ion transmembrane transport / calcium channel activity / calcium ion transport / calmodulin binding / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily V member 6 / Transient receptor potential cation channel subfamily V member 5/6 / Ankyrin repeat-containing domain / 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 ...Transient receptor potential cation channel subfamily V member 6 / Transient receptor potential cation channel subfamily V member 5/6 / Ankyrin repeat-containing domain / 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 / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Ion transport domain / Ion transport protein / Alpha Horseshoe / Mainly Alpha
Similarity search - Domain/homology
Chem-81F / Transient receptor potential cation channel subfamily V member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsNeuberger, A. / Nadezhdin, K.D. / Singh, A.K. / Sobolevsky, A.I.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA206573 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS083660 United States
National Science Foundation (NSF, United States)1818086 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM124165 United States
CitationJournal: Sci Adv / Year: 2020
Title: Inactivation-mimicking block of the epithelial calcium channel TRPV6.
Authors: Rajesh Bhardwaj / Sonja Lindinger / Arthur Neuberger / Kirill D Nadezhdin / Appu K Singh / Micael R Cunha / Isabella Derler / Gergely Gyimesi / Jean-Louis Reymond / Matthias A Hediger / ...Authors: Rajesh Bhardwaj / Sonja Lindinger / Arthur Neuberger / Kirill D Nadezhdin / Appu K Singh / Micael R Cunha / Isabella Derler / Gergely Gyimesi / Jean-Louis Reymond / Matthias A Hediger / Christoph Romanin / Alexander I Sobolevsky /
Abstract: Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of ...Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.
History
DepositionSep 15, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 9, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 28, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 6
B: Transient receptor potential cation channel subfamily V member 6
C: Transient receptor potential cation channel subfamily V member 6
D: Transient receptor potential cation channel subfamily V member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)310,23911
Polymers308,0874
Non-polymers2,1527
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29190 Å2
ΔGint-262 kcal/mol
Surface area120090 Å2

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 6 / TrpV6 / CaT-like / CaT-L / Calcium transport protein 1 / CaT1 / Epithelial calcium channel 2 / ECaC2


Mass: 77021.805 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPV6, ECAC2 / Production host: Homo sapiens (human) / References: UniProt: Q9H1D0
#2: Chemical
ChemComp-81F / 1-(5-bromopyridin-3-yl)-4-[cis-4-(3-methylphenyl)cyclohexyl]piperazine


Mass: 414.382 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C22H28BrN3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestY
Has protein modificationN

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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: sample 1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethane1
30.01 %glyco-diosgenin (GDN)1
40.001 %Cholesteryl hemisuccinate1
51 mM(4- phenylcyclohexyl)piperazine inhibitor Br-cis-22a1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 3 sec. / Electron dose: 70.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5426
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2.14particle selection
4cryoSPARC2.14CTF correction
8PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1515725
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187030 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00820209
ELECTRON MICROSCOPYf_angle_d1.05527424
ELECTRON MICROSCOPYf_dihedral_angle_d14.44912043
ELECTRON MICROSCOPYf_chiral_restr0.0583107
ELECTRON MICROSCOPYf_plane_restr0.0073454

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