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- PDB-7d2k: Crystal structure of rat TRPV6 in complex with (4- phenylcyclohex... -

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Entry
Database: PDB / ID: 7d2k
TitleCrystal structure of rat TRPV6 in complex with (4- phenylcyclohexyl)piperazine inhibitor Br-cis-22a
ComponentsTransient receptor potential cation channel subfamily V member 6
KeywordsTRANSPORT PROTEIN/INHIBITOR / Ion channels / TRP channels / Membrane proteins / TRANSPORT PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


parathyroid hormone secretion / TRP channels / calcium-activated cation channel activity / calcium ion import / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / response to calcium ion ...parathyroid hormone secretion / TRP channels / calcium-activated cation channel activity / calcium ion import / calcium ion import across plasma membrane / calcium ion homeostasis / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / response to calcium ion / 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 6 / Transient receptor potential cation channel subfamily V member 5/6 / Transient receptor potential cation channel subfamily V / 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-GQC / Transient receptor potential cation channel subfamily V member 6
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.698 Å
AuthorsSingh, A.K. / Neuberger, A. / Nadezhdin, K.D. / Sobolevsky, A.I.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA206573 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 16, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 20, 2021Group: Database references / Structure summary / Category: audit_author / citation_author / Item: _audit_author.name / _citation_author.name
Revision 1.2Apr 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)78,0044
Polymers77,1331
Non-polymers8713
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)146.228, 146.228, 114.648
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number90
Space group name H-MP4212
Components on special symmetry positions
IDModelComponents
11A-701-

CA

21A-703-

GQC

31A-703-

GQC

41A-703-

GQC

51A-703-

GQC

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Components

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


Mass: 77133.492 Da / Num. of mol.: 1 / Fragment: UNP residues 41-709 / Mutation: I62Y, L132N, M96Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Trpv6 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q9R186
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-GQC / 1-(5-bromanylpyridin-3-yl)-4-[4-(3-methylphenyl)cyclohexyl]piperazin-4-ium


Mass: 415.390 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H29BrN3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.94 Å3/Da / Density % sol: 68.78 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 150 mM nacl, 100 mM TRIS, 22% PEG 350 / PH range: 8.0-8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.92 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Mar 12, 2019
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
ReflectionResolution: 3.6→47.129 Å / Num. obs: 14416 / % possible obs: 99.12 % / Redundancy: 8.13 % / CC1/2: 0.99 / Net I/σ(I): 1.45
Reflection shellResolution: 3.6→3.9 Å / Num. unique obs: 2587 / CC1/2: 0.61 / % possible all: 96.2

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5WO7

5wo7


Resolution: 3.698→47.12 Å / SU ML: 0.76 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 48.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3274 690 5.01 %
Rwork0.3162 --
obs0.3168 13778 99.79 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.698→47.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4819 0 53 0 4872
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0024960
X-RAY DIFFRACTIONf_angle_d0.5246739
X-RAY DIFFRACTIONf_dihedral_angle_d14.2332941
X-RAY DIFFRACTIONf_chiral_restr0.037768
X-RAY DIFFRACTIONf_plane_restr0.004849
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.698-3.98330.45321340.42182528X-RAY DIFFRACTION99
3.9833-4.38390.39141350.38262571X-RAY DIFFRACTION100
4.3839-5.01770.37891360.32492598X-RAY DIFFRACTION100
5.0177-6.31930.31971380.33852620X-RAY DIFFRACTION100
6.3193-47.120.29031470.28182771X-RAY DIFFRACTION100

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