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- PDB-9mfl: Structure of zebrafish OTOP1 in nanodisc in the presence of inhib... -

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Basic information

Entry
Database: PDB / ID: 9mfl
TitleStructure of zebrafish OTOP1 in nanodisc in the presence of inhibitor C11
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
(3beta,5beta,14beta,17alpha)-cholestan-3-ol / CHOLESTEROL HEMISUCCINATE / Proton channel OTOP1
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)135,13610
Polymers131,6342
Non-polymers3,5028
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-QNJ / (3beta,5beta,14beta,17alpha)-cholestan-3-ol


Mass: 388.669 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H48O
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: 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
41 %dimethylsulfoxide1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: sample was complexed with small molecule C11, reaching a final concentration of 1.11mM, and 1% 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

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 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: 9.8 sec. / Electron dose: 50.37 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3552
Details: Forty-nine frames of 200 ms exposure time were collected per movie
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 49

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.0particle selectiontemplate picker
2Leginonimage acquisition
4Gctf1.06CTF correction
7Coot0.9.8.7model fitting
9Coot0.9.8.7model refinement
10PHENIX1.20.1_4487model refinement
11Rosetta2022.11model refinement
12cryoSPARC3.3.0initial Euler assignment
13cryoSPARC3.3.0final Euler assignment
14cryoSPARC3.3.0classification
15cryoSPARC3.3.03D reconstruction
Image processingDetails: Micrographs with CTF estimates (Gctf) worse than 5A were discarded.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3410686
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164766
Details: final reported resolution of 3.73A 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
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066010
ELECTRON MICROSCOPYf_angle_d1.0058200
ELECTRON MICROSCOPYf_dihedral_angle_d13.6432056
ELECTRON MICROSCOPYf_chiral_restr0.094980
ELECTRON MICROSCOPYf_plane_restr0.019934

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