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- PDB-6d7t: Cryo-EM structure of human TRPV6-Y467A in complex with 2-Aminoeth... -

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Basic information

Entry
Database: PDB / ID: 6d7t
TitleCryo-EM structure of human TRPV6-Y467A in complex with 2-Aminoethoxydiphenyl borate (2-APB)
ComponentsTransient receptor potential cation channel subfamily V member 6
KeywordsMEMBRANE PROTEIN / Ion channels / transporter / calcium channel / epithelial calcium channel
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 / calcium ion transmembrane transport / calcium channel activity / response to calcium ion / 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 / calcium ion transmembrane transport / calcium channel activity / response to calcium ion / calcium ion transport / calmodulin binding / 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 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 6 / 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
2-aminoethyl diphenylborinate / Transient receptor potential cation channel subfamily V member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.44 Å
AuthorsSingh, A.K. / Saotome, K. / McGoldrick, L.L. / Sobolevsky, A.I.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)1R01CA206573-01A1 United States
CitationJournal: Nat Commun / Year: 2018
Title: Structural bases of TRP channel TRPV6 allosteric modulation by 2-APB.
Authors: Appu K Singh / Kei Saotome / Luke L McGoldrick / Alexander I Sobolevsky /
Abstract: Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its ...Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its expression is dramatically increased in numerous types of cancer. TRPV6 inhibitors suppress tumor growth, but the molecular mechanism of inhibition remains unknown. Here, we present crystal and cryo-EM structures of human and rat TRPV6 bound to 2-aminoethoxydiphenyl borate (2-APB), a TRPV6 inhibitor and modulator of numerous TRP channels. 2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helix bundle. Comparing human wild-type and high-affinity mutant Y467A structures, we show that 2-APB induces TRPV6 channel closure by modulating protein-lipid interactions. Mutagenesis and functional analyses suggest that the identified 2-APB binding site might be present in other members of vanilloid subfamily TRP channels. Our findings reveal a mechanism of ion channel allosteric modulation that can be exploited for therapeutic design.
History
DepositionApr 25, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 18, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization

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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)341,28810
Polymers340,3074
Non-polymers9816
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 85076.859 Da / Num. of mol.: 4 / Mutation: Y467A
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-FZ4 / 2-aminoethyl diphenylborinate / 2-Aminoethoxydiphenyl borate


Mass: 225.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H16BNO
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

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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: TRPV6 in amphipols / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMTRISTRIS1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2MotionCorr2particle selection
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77460 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00719452
ELECTRON MICROSCOPYf_angle_d1.08126396
ELECTRON MICROSCOPYf_dihedral_angle_d12.56411676
ELECTRON MICROSCOPYf_chiral_restr0.0563012
ELECTRON MICROSCOPYf_plane_restr0.0073332

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