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- PDB-7mxo: CryoEM structure of human NKCC1 -

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Basic information

Entry
Database: PDB / ID: 7mxo
TitleCryoEM structure of human NKCC1
ComponentsSolute carrier family 12 member 2
KeywordsMEMBRANE PROTEIN / Sodium / potassium / chloride / co-transporter / ions / human / CTD / TMD / full-length
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / metal ion transmembrane transporter activity / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity ...positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / cell body membrane / metal ion transmembrane transporter activity / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / potassium ion transmembrane transporter activity / transepithelial chloride transport / intracellular chloride ion homeostasis / ammonium transmembrane transport / negative regulation of vascular wound healing / sodium ion homeostasis / ammonium channel activity / chloride ion homeostasis / cell projection membrane / cellular response to chemokine / potassium ion homeostasis / T cell chemotaxis / intracellular sodium ion homeostasis / sodium ion import across plasma membrane / cell volume homeostasis / cellular response to potassium ion / hyperosmotic response / regulation of spontaneous synaptic transmission / gamma-aminobutyric acid signaling pathway / maintenance of blood-brain barrier / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / lateral plasma membrane / transport across blood-brain barrier / monoatomic ion transport / sodium ion transmembrane transport / basal plasma membrane / cytoplasmic vesicle membrane / chloride transmembrane transport / cell periphery / cell projection / Hsp90 protein binding / extracellular vesicle / protein-folding chaperone binding / cell body / basolateral plasma membrane / neuron projection / apical plasma membrane / neuronal cell body / protein kinase binding / extracellular exosome / membrane / plasma membrane
Similarity search - Function
Amino acid/polyamine transporter I / Amino acid/polyamine transporter I / Solute carrier family 12 member 1/2 / Solute carrier family 12 member 2 / 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/polyamine transporter I / Amino acid/polyamine transporter I / Solute carrier family 12 member 1/2 / Solute carrier family 12 member 2 / 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 / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
: / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsMoseng, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)RO1 AI145069 United States
CitationJournal: Sci Adv / Year: 2022
Title: Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling.
Authors: Mitchell A Moseng / Chih-Chia Su / Kerri Rios / Meng Cui / Meinan Lyu / Przemyslaw Glaza / Philip A Klenotic / Eric Delpire / Edward W Yu /
Abstract: The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na-dependent transporter responsible for simultaneously translocating Na, K, and Cl ions into cells. In human tissue, NKCC1 plays a critical ...The Na-K-2Cl cotransporter-1 (NKCC1) is an electroneutral Na-dependent transporter responsible for simultaneously translocating Na, K, and Cl ions into cells. In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Here, we report four structures of human NKCC1 (hNKCC1), both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryo-electron microscopy. These structures allow us to directly observe various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. Together, our findings enable us to delineate an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1.
History
DepositionMay 19, 2021Deposition site: RCSB / Processing site: RCSB
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Revision 1.2Apr 12, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3May 31, 2023Group: Data collection / Category: em_image_recording / Item: _em_image_recording.film_or_detector_model
Revision 1.4Oct 16, 2024Group: Data collection / Structure summary
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Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Solute carrier family 12 member 2
B: Solute carrier family 12 member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,1866
Polymers205,0372
Non-polymers1494
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Solute carrier family 12 member 2 / Basolateral Na-K-Cl symporter / Bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2


Mass: 102518.414 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293 / References: UniProt: P55011
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
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: Full-length human NKCC1 with NaCl and KCl / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: .131 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc1_3777: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 354623 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313303
ELECTRON MICROSCOPYf_angle_d0.83718036
ELECTRON MICROSCOPYf_dihedral_angle_d24.5041776
ELECTRON MICROSCOPYf_chiral_restr0.2242087
ELECTRON MICROSCOPYf_plane_restr0.0032242

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