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- PDB-8gqu: AK-42 inhibitor binding human ClC-2 TMD -

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Basic information

Entry
Database: PDB / ID: 8gqu
TitleAK-42 inhibitor binding human ClC-2 TMD
ComponentsChloride channel protein 2
KeywordsMEMBRANE PROTEIN / homo-dimer
Function / homology
Function and homology information


regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / astrocyte end-foot / stabilization of membrane potential / acinar cell differentiation / cellular hypotonic response / voltage-gated chloride channel activity / regulation of resting membrane potential ...regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / astrocyte end-foot / stabilization of membrane potential / acinar cell differentiation / cellular hypotonic response / voltage-gated chloride channel activity / regulation of resting membrane potential / axon initial segment / chloride channel regulator activity / dendritic spine membrane / chloride transport / phagocytosis, engulfment / positive regulation of oligodendrocyte differentiation / chloride channel complex / lung development / Stimuli-sensing channels / myelin sheath / retina development in camera-type eye / perikaryon / basolateral plasma membrane / postsynaptic membrane / plasma membrane
Similarity search - Function
Chloride channel ClC-2 / : / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / CBS domain profile.
Similarity search - Domain/homology
Chem-GH6 / Chloride channel protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWang, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42.
Authors: Tao Ma / Lei Wang / Anping Chai / Chao Liu / Wenqiang Cui / Shuguang Yuan / Shannon Wing Ngor Au / Liang Sun / Xiaokang Zhang / Zhenzhen Zhang / Jianping Lu / Yuanzhu Gao / Peiyi Wang / ...Authors: Tao Ma / Lei Wang / Anping Chai / Chao Liu / Wenqiang Cui / Shuguang Yuan / Shannon Wing Ngor Au / Liang Sun / Xiaokang Zhang / Zhenzhen Zhang / Jianping Lu / Yuanzhu Gao / Peiyi Wang / Zhifang Li / Yujie Liang / Horst Vogel / Yu Tian Wang / Daping Wang / Kaige Yan / Huawei Zhang /
Abstract: ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK- ...ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.
History
DepositionAug 30, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 8, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chloride channel protein 2
B: Chloride channel protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,0634
Polymers197,2852
Non-polymers7782
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Chloride channel protein 2 / ClC-2


Mass: 98642.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLCN2 / Production host: Homo sapiens (human) / Strain (production host): HEK293F / References: UniProt: P51788
#2: Chemical ChemComp-GH6 / 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid


Mass: 389.232 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H14Cl2N2O3 / Feature type: SUBJECT OF INVESTIGATION
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: AK-42 inhibitor binding human ClC-2 TMD 3.5A / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 180 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44153 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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