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- PDB-8vpp: Phosphorylated human NCC in complex with chlorthalidone -

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Basic information

Entry
Database: PDB / ID: 8vpp
TitlePhosphorylated human NCC in complex with chlorthalidone
ComponentsSolute carrier family 12 member 3
KeywordsTRANSPORT PROTEIN / Sodium chloride cotransporter / phosphorylation / thiazide-like diuretics / chlorthalidone
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 / SLC12A transporter family / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
Chem-KLT / Solute carrier family 12 member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhao, Y.X. / Cao, E.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK128592 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
DepositionJan 16, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.0Sep 4, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Sep 4, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0Sep 4, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 4, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 4, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 4, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 23, 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 14, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 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)227,0683
Polymers226,7292
Non-polymers3391
Water362
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: 113364.648 Da / Num. of mol.: 2
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-KLT / 2-chloro-5-[(1S)-1-hydroxy-3-oxo-2H-isoindol-1-yl]benzenesulfonamide / chlorthalidone


Mass: 338.766 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H11ClN2O4S
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / 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 human NCC in complex with chlorthalidone
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: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: DIFFRACTION / Nominal defocus max: 3500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 40 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: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2851886 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410163
ELECTRON MICROSCOPYf_angle_d1.05513808
ELECTRON MICROSCOPYf_dihedral_angle_d9.3891399
ELECTRON MICROSCOPYf_chiral_restr0.041555
ELECTRON MICROSCOPYf_plane_restr0.0071742

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