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- PDB-9bwt: human sodium chloride cotransporter NCC S344E in the phosphorylat... -

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Basic information

Entry
Database: PDB / ID: 9bwt
Titlehuman sodium chloride cotransporter NCC S344E in the phosphorylation state and in complex with hydrochlorothiazide
ComponentsSolute carrier family 12 member 3
KeywordsTRANSPORT PROTEIN / sodium chloride cotransporter / phosphorylation / thiazide diuretics
Function / homology
Function and homology information


sodium:chloride symporter activity / apical plasma membrane
Similarity search - Function
Thiazide-sensitive Na-K-Cl co-transporter / Amino acid permease, N-terminal / Amino acid permease N-terminal / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Chem-HCZ / Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsCao, E.H. / Zhao, Y.X.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)5R01DK128592 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural bases for Na-Cl cotransporter inhibition by thiazide diuretic drugs and activation by kinases.
Authors: Yongxiang Zhao / Heidi Schubert / Alan Blakely / Biff Forbush / Micholas Dean Smith / Jesse Rinehart / Erhu Cao /
Abstract: The Na-Cl cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated ...The Na-Cl cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.
History
DepositionMay 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
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Revision 1.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Solute carrier family 12 member 3
B: Solute carrier family 12 member 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,1255
Polymers226,8132
Non-polymers1,3123
Water1086
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Solute carrier family 12 member 3


Mass: 113406.680 Da / Num. of mol.: 2 / Mutation: S344E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A3 / Production host: Homo sapiens (human) / References: UniProt: J3QSS1
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: Chemical ChemComp-HCZ / 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide


Mass: 297.739 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H8ClN3O4S2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: Phosphorylated SLC12A3 transporter in complex with hydrochlorothiazide
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Human embryonic kidney 293
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 4 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 FIELD / Nominal defocus max: 3500 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

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3281696 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410183
ELECTRON MICROSCOPYf_angle_d0.48513863
ELECTRON MICROSCOPYf_dihedral_angle_d6.5841444
ELECTRON MICROSCOPYf_chiral_restr0.0391557
ELECTRON MICROSCOPYf_plane_restr0.0041740

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