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- PDB-8dhm: Human TMEM175 in complex with 4-aminopyridine -

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Basic information

Entry
Database: PDB / ID: 8dhm
TitleHuman TMEM175 in complex with 4-aminopyridine
ComponentsEndosomal/lysosomal potassium channel TMEM175
KeywordsTRANSPORT PROTEIN / potassium channel / ion channel
Function / homology
Function and homology information


lysosomal lumen pH elevation / phagosome-lysosome fusion / regulation of lysosomal lumen pH / proton channel activity / potassium ion leak channel activity / arachidonic acid binding / potassium channel activity / potassium ion transmembrane transport / proton transmembrane transport / neuron cellular homeostasis ...lysosomal lumen pH elevation / phagosome-lysosome fusion / regulation of lysosomal lumen pH / proton channel activity / potassium ion leak channel activity / arachidonic acid binding / potassium channel activity / potassium ion transmembrane transport / proton transmembrane transport / neuron cellular homeostasis / lysosome / endosome membrane / endosome / lysosomal membrane
Similarity search - Function
Endosomal/lysomomal potassium channel TMEM175 / Endosomal/lysosomal potassium channel TMEM175
Similarity search - Domain/homology
4-AMINOPYRIDINE / : / Endosomal/lysosomal proton channel TMEM175
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsOh, S. / Hite, R.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141553 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175.
Authors: SeCheol Oh / Robyn Stix / Wenchang Zhou / José D Faraldo-Gómez / Richard K Hite /
Abstract: Transmembrane protein 175 (TMEM175) is an evolutionarily distinct lysosomal cation channel whose mutation is associated with the development of Parkinson's disease. Here, we present a cryoelectron ...Transmembrane protein 175 (TMEM175) is an evolutionarily distinct lysosomal cation channel whose mutation is associated with the development of Parkinson's disease. Here, we present a cryoelectron microscopy structure and molecular simulations of TMEM175 bound to 4-aminopyridine (4-AP), the only known small-molecule inhibitor of TMEM175 and a broad K channel inhibitor, as well as a drug approved by the Food and Drug Administration against multiple sclerosis. The structure shows that 4-AP, whose mode of action had not been previously visualized, binds near the center of the ion conduction pathway, in the open state of the channel. Molecular dynamics simulations reveal that this binding site is near the middle of the transmembrane potential gradient, providing a rationale for the voltage-dependent dissociation of 4-AP from TMEM175. Interestingly, bound 4-AP rapidly switches between three predominant binding poses, stabilized by alternate interaction patterns dictated by the twofold symmetry of the channel. Despite this highly dynamic binding mode, bound 4-AP prevents not only ion permeation but also water flow. Together, these studies provide a framework for the rational design of novel small-molecule inhibitors of TMEM175 that might reveal the role of this channel in human lysosomal physiology both in health and disease.
History
DepositionJun 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Endosomal/lysosomal potassium channel TMEM175
A: Endosomal/lysosomal potassium channel TMEM175
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,5867
Polymers111,3342
Non-polymers2525
Water1,63991
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Endosomal/lysosomal potassium channel TMEM175


Mass: 55667.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TMEM175 / Production host: Homo sapiens (human) / References: UniProt: Q9BSA9
#2: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-4AP / 4-AMINOPYRIDINE / 4-Aminopyridine


Mass: 95.122 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H7N2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 91 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Human TMEM175 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMPotassium chlorideKCl1
250 mMTris-HClTris1
30.1 %Lauryl maltose neopentyl glycol1
42 mMDithiothreitol1
51 %Dimethyl sulfoxide1
61 mM4-aminopyridine1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 61 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1.2particle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4153614
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 261536 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Details: Real Space Refinement
Atomic model buildingPDB-ID: 6WC9

6wc9


Accession code: 6WC9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0065837
ELECTRON MICROSCOPYf_angle_d0.7377954
ELECTRON MICROSCOPYf_dihedral_angle_d4.426780
ELECTRON MICROSCOPYf_chiral_restr0.042978
ELECTRON MICROSCOPYf_plane_restr0.011975

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