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- PDB-9mfm: Structure of zebrafish OTOP1 in nanodisc in complex with inhibito... -

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Basic information

Entry
Database: PDB / ID: 9mfm
TitleStructure of zebrafish OTOP1 in nanodisc in complex with inhibitor C2.36
ComponentsProton channel OTOP1
KeywordsMEMBRANE PROTEIN / proton channel / ion channel / OTOP / Otopetrin
Function / homology
Function and homology information


otolith formation / otolith mineralization / proton channel activity / inner ear morphogenesis / cholesterol binding / negative regulation of type II interferon-mediated signaling pathway / microvillus / proton transmembrane transport / cellular response to insulin stimulus / identical protein binding ...otolith formation / otolith mineralization / proton channel activity / inner ear morphogenesis / cholesterol binding / negative regulation of type II interferon-mediated signaling pathway / microvillus / proton transmembrane transport / cellular response to insulin stimulus / identical protein binding / membrane / plasma membrane
Similarity search - Function
: / CHOLESTEROL / CHOLESTEROL HEMISUCCINATE / Proton channel OTOP1
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsBurendei, B. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: Nat Commun / Year: 2025
Title: Structure-guided discovery of Otopetrin 1 inhibitors reveals druggable binding sites at the intrasubunit interface.
Authors: Batuujin Burendei / Joshua P Kaplan / Gerardo M Orellana / Emily R Liman / Stefano Forli / Andrew B Ward /
Abstract: Proton conductance across cell membranes serves many biological functions, ranging from the regulation of intracellular and extracellular pH to the generation of electrical signals that lead to ...Proton conductance across cell membranes serves many biological functions, ranging from the regulation of intracellular and extracellular pH to the generation of electrical signals that lead to sour taste perception. Otopetrins (OTOPs) are a conserved, eukaryotic family of proton-selective ion channels, one of which (OTOP1) serves as a gustatory sensor for sour tastes and ammonium chloride. As the functional properties and structures of OTOP channels were only recently described, there are presently few tools available to modulate their activity. Here, we perform subsequent rounds of molecular docking-based virtual screening against the structure of zebrafish OTOP1, followed by functional testing using whole-cell patch-clamp electrophysiology, and identify several small molecule inhibitors that are effective in the low-to-mid µM range. Cryo-electron microscopy structures reveal inhibitor binding sites in the intrasubunit interface that are validated by functional testing of mutant channels. Our findings reveal pockets that can be targeted for small molecule discovery to develop modulators for Otopetrins. Such modulators can serve as useful toolkit molecules for future investigations of structure-function relationships or physiological roles of Otopetrins.
History
DepositionDec 10, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proton channel OTOP1
B: Proton channel OTOP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,03014
Polymers131,6342
Non-polymers4,39612
Water00
1


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

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Components

#1: Protein Proton channel OTOP1 / Otopetrin-1


Mass: 65817.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: first two residues (GP) are leftover residues from a 3C protease cleavage site. 3rd residue (V) corresponds to the 2nd residue of the zebrafish OTOP1 sequence (Uniprot:Q7ZWK8)
Source: (gene. exp.) Danio rerio (zebrafish) / Gene: otop1 / Plasmid: pEG / Details (production host): BacMam expression plasmid / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q7ZWK8
#2: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical
ChemComp-A1BKT / 4-[(4R)-5H,11H-[1,2,4]triazolo[3,4-c][1,4]benzoxazepin-3-yl]phenol


Mass: 279.293 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C16H13N3O2 / Feature type: SUBJECT OF INVESTIGATION
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: zebrafish OTOP1 in MSP2N2 nanodisc / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.131 MDa / Experimental value: NO
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F / Plasmid: pEG
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
125 mMTris1
2150 mMNaCl1
32 mMdithiothreitol1
45 %dimethylsulfoxide1
SpecimenConc.: 5.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: sample was complexed with small molecule C2.36, reaching a final concentration of 1 mM, and 5% DMSO
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 45 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 26378
Image scansWidth: 4096 / Height: 4096

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
Image processingDetails: Micrographs with CTF estimates (cryoSPARC Live) worse than 5A were discarded.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7236014
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 290491
Details: final reported resolution of 3.42A was obtained through the Remote 3DFSC Processing server
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6NF4

6nf4


Accession code: 6NF4 / Source name: PDB / Type: experimental model

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