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- PDB-9dyo: BEST2 + PABA closed state -

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Basic information

Entry
Database: PDB / ID: 9dyo
TitleBEST2 + PABA closed state
ComponentsBestrophin-2a
KeywordsMEMBRANE PROTEIN / calcium-activated chloride channel / para-aminobenzoic acid (PABA)-bound anion channel / channel-activator complex
Function / homology
Function and homology information


intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / Stimuli-sensing channels / sensory perception of smell / basolateral plasma membrane ...intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / Stimuli-sensing channels / sensory perception of smell / basolateral plasma membrane / cilium / metal ion binding / plasma membrane
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.57 Å
AuthorsOwji, A.P. / Kittredge, A. / Zhang, Y. / Yang, T.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM149252 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127652 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY028758 United States
Other privateIrma T. Hirschl Trust CU20-4313 United States
Other privateResearch to Prevent Blindness (RPB) CU22-1892 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129539 United States
CitationJournal: Nat Commun / Year: 2024
Title: Neurotransmitter-bound bestrophin channel structures reveal small molecule drug targeting sites for disease treatment.
Authors: Aaron P Owji / Jingyun Dong / Alec Kittredge / Jiali Wang / Yu Zhang / Tingting Yang /
Abstract: Best1 and Best2 are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. Here, ...Best1 and Best2 are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. Here, we solved glutamate- and γ-aminobutyric acid (GABA)-bound Best2 structures, which delineate an intracellular glutamate binding site and an extracellular GABA binding site on Best2, respectively, identified extracellular GABA as a permeable activator of Best2, and elucidated the co-regulation of Best2 by glutamate, GABA and glutamine synthetase in vivo. We further identified multiple small molecules as activators of the bestrophin channels. Extensive analyses were carried out for a potent activator, 4-aminobenzoic acid (PABA): PABA-bound Best1 and Best2 structures are solved and illustrate the same binding site as in GABA-bound Best2; PABA treatment rescues the functional deficiency of patient-derived Best1 mutations. Together, our results demonstrate the mechanism and potential of multiple small molecule candidates as clinically applicable drugs for bestrophin-associated diseases/conditions.
History
DepositionOct 14, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Bestrophin-2a
C: Bestrophin-2a
E: Bestrophin-2a
A: Bestrophin-2a
B: Bestrophin-2a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)286,17911
Polymers285,9435
Non-polymers2366
Water00
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
Bestrophin-2a / Vitelliform macular dystrophy 2-like protein 1


Mass: 57188.637 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BEST2, VMD2L1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8NFU1
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
Has ligand of interestN
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: BEST2 + PABA closed state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.286 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
240 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
30.008 % w/vglyco-diosgeninGDN1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R0./1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS 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: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1051
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1PHENIX1.20.1_4487:model refinement
2Leginonimage acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 194813
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26692 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 8D1E

8d1e


Pdb chain-ID: A / Accession code: 8D1E / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00217060
ELECTRON MICROSCOPYf_angle_d0.42722885
ELECTRON MICROSCOPYf_dihedral_angle_d13.6952915
ELECTRON MICROSCOPYf_chiral_restr0.0342405
ELECTRON MICROSCOPYf_plane_restr0.0032745

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