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- PDB-7zgo: Cryo-EM structure of human NKCC1 (TM domain) -

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Basic information

Entry
Database: PDB / ID: 7zgo
TitleCryo-EM structure of human NKCC1 (TM domain)
ComponentsSolute carrier family 12 member 2
KeywordsTRANSPORT PROTEIN / SLC12 family / NKCC1 in complex with Na+ / K+ and 2Cl- / LeuT-fold
Function / homology
Function and homology information


positive regulation of cell volume / positive regulation of aspartate secretion / transepithelial ammonium transport / regulation of matrix metallopeptidase secretion / metal ion transmembrane transporter activity / cell body membrane / 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 / metal ion transmembrane transporter activity / cell body membrane / inorganic anion import across plasma membrane / inorganic cation import across plasma membrane / chloride:monoatomic cation symporter activity / sodium:potassium:chloride symporter activity / transepithelial chloride transport / potassium ion transmembrane transporter activity / Cation-coupled Chloride cotransporters / ammonium transmembrane transport / sodium ion homeostasis / intracellular chloride ion homeostasis / negative regulation of vascular wound healing / chloride ion homeostasis / cellular response to potassium ion / cell projection membrane / ammonium channel activity / intracellular potassium ion homeostasis / potassium ion homeostasis / T cell chemotaxis / cellular response to chemokine / sodium ion import across plasma membrane / intracellular sodium ion homeostasis / hyperosmotic response / cell volume homeostasis / gamma-aminobutyric acid signaling pathway / regulation of spontaneous synaptic transmission / maintenance of blood-brain barrier / potassium ion import across plasma membrane / transport across blood-brain barrier / lateral plasma membrane / sodium ion transmembrane transport / monoatomic ion transport / chloride transmembrane transport / basal plasma membrane / cell projection / cell periphery / Hsp90 protein binding / cytoplasmic vesicle membrane / 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
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 family / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
: / Chem-POV / CHOLESTEROL HEMISUCCINATE / Solute carrier family 12 member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsNissen, P. / Fenton, R. / Neumann, C. / Lindtoft Rosenbaek, L. / Kock Flygaard, R. / Habeck, M. / Lykkegaard Karlsen, J. / Wang, Y. / Lindorff-Larsen, K. / Gad, H. ...Nissen, P. / Fenton, R. / Neumann, C. / Lindtoft Rosenbaek, L. / Kock Flygaard, R. / Habeck, M. / Lykkegaard Karlsen, J. / Wang, Y. / Lindorff-Larsen, K. / Gad, H. / Hartmann, R. / Lyons, J.
Funding support Denmark, France, 6items
OrganizationGrant numberCountry
Lundbeckfonden2015-3225 Denmark
LundbeckfondenR310-2018-3713 Denmark
Leducq Foundation17CVD05 France
Novo Nordisk FoundationNNF21OC0067647 Denmark
Novo Nordisk FoundationNNF17OC0029724 Denmark
Novo Nordisk FoundationNNF19OC0058439 Denmark
CitationJournal: EMBO J / Year: 2022
Title: Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.
Authors: Caroline Neumann / Lena Lindtoft Rosenbaek / Rasmus Kock Flygaard / Michael Habeck / Jesper Lykkegaard Karlsen / Yong Wang / Kresten Lindorff-Larsen / Hans Henrik Gad / Rune Hartmann / ...Authors: Caroline Neumann / Lena Lindtoft Rosenbaek / Rasmus Kock Flygaard / Michael Habeck / Jesper Lykkegaard Karlsen / Yong Wang / Kresten Lindorff-Larsen / Hans Henrik Gad / Rune Hartmann / Joseph Anthony Lyons / Robert A Fenton / Poul Nissen /
Abstract: The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na -dependent Cl - and K -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, ...The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na -dependent Cl - and K -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na , K , and 2 Cl ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl binding at the Cl1 site together with the nearby K ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.
History
DepositionApr 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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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)272,14623
Polymers263,6392
Non-polymers8,50721
Water43224
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4940 Å2
ΔGint-82 kcal/mol
Surface area38300 Å2

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Components

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Protein , 1 types, 2 molecules AB

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


Mass: 131819.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A2, NKCC1 / Production host: Homo sapiens (human) / References: UniProt: P55011

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Non-polymers , 6 types, 45 molecules

#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / 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
#4: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#6: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Dimeric NKCC1 in complex with Na+, K+ and 2Cl-COMPLEX#10RECOMBINANT
2NKCC1 in complex with Na+, K+ and 2Cl-COMPLEX#11RECOMBINANT
3NKCC1 in complex with Na+, K+ and 2Cl-COMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.263366 MDaNO
220.131683 MDaNO
330.131683 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
22Homo sapiens (human)9606
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Buffer solutionpH: 8
Details: 0.01% glycol-diosgenin (GDN, Anatrace), 200mM NaCl, 200mM KCl, 20mM Tris-HCl pH 8.0 and 0.07mM bumetanide
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: concentration (mg/mL): 0.9-1.5
Specimen supportDetails: GloQube (Quorum) 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Details: 3 uL sample was applied to the gold foil side of the grid and blotted iusing a blot force of 0 and blot time of 3-4 seconds before plunge- freezing into liquid ethane cooled by liquid nitrogen.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELD / Specimen-ID: 1

IDNominal defocus max (nm)Nominal defocus min (nm)Nominal magnification (X)
11600600165000
22400500130000
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of real images
1140.12GATAN K3 BIOQUANTUM (6k x 4k)10063
2130.78GATAN K3 BIOQUANTUM (6k x 4k)9122
3260.438GATAN K3 BIOQUANTUM (6k x 4k)6190
EM imaging optics
Energyfilter nameIDImaging-IDEnergyfilter slit width (eV)
GIF Bioquantum1120
GIF Bioquantum2220

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Processing

EM software
IDNameVersionCategoryDetails
4RELION3.1CTF correctionCTFFind4
7PHENIXmodel fitting
10PHENIXmodel refinement
14RELION3.13D reconstruction
15cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1002976
Details: Dataset 1: 1002976 particles Dataset 2: 1566691 Dataset 3: 2710603
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 258791 / Symmetry type: POINT
Atomic model buildingB value: 61.44 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6NPL

6npl


Pdb chain-ID: A

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