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- EMDB-11803: Structure of Human Potassium Chloride Transporter KCC1 in NaCl (S... -

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Basic information

Entry
Database: EMDB / ID: EMD-11803
TitleStructure of Human Potassium Chloride Transporter KCC1 in NaCl (Subclass 2)
Map data
Sample
  • Complex: Homodimeric complex of human potassium chloride transporter KCC1 in complex with ATP and magnesium
    • Protein or peptide: Solute carrier family 12 member 4
  • Protein or peptide: Solute carrier family 12 member 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / chloride transmembrane transport / potassium ion transmembrane transport / chemical synaptic transmission ...potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / chloride transmembrane transport / potassium ion transmembrane transport / chemical synaptic transmission / lysosomal membrane / synapse / protein kinase binding / ATP binding / membrane / plasma membrane
Similarity search - Function
K/Cl co-transporter 1 / K/Cl co-transporter / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsEbenhoch R / Chi G / Man H / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R / Moreau C / Snee M ...Ebenhoch R / Chi G / Man H / Wang D / McKinley G / Mukhopadhyay SMM / MacLean EM / Chalk R / Moreau C / Snee M / Bohstedt T / Singh NK / Abrusci P / Liko I / Tehan BG / Almeida FG / Arrowsmith CH / Tang H / Robinson CV / Bountra C / Edwards AM / Marsden BD / Burgess-Brown NA / Duerr KL / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
European Commission United Kingdom
CitationJournal: EMBO J / Year: 2021
Title: Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.
Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong ...Authors: Gamma Chi / Rebecca Ebenhoch / Henry Man / Haiping Tang / Laurence E Tremblay / Gabriella Reggiano / Xingyu Qiu / Tina Bohstedt / Idlir Liko / Fernando G Almeida / Alexandre P Garneau / Dong Wang / Gavin McKinley / Christophe P Moreau / Kiran D Bountra / Patrizia Abrusci / Shubhashish M M Mukhopadhyay / Alejandra Fernandez-Cid / Samira Slimani / Julie L Lavoie / Nicola A Burgess-Brown / Ben Tehan / Frank DiMaio / Ali Jazayeri / Paul Isenring / Carol V Robinson / Katharina L Dürr /
Abstract: Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions ...Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
History
DepositionSep 28, 2020-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.21
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.21
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7air
  • Surface level: 0.21
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11803.png

Downloads & links

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Map

FileDownload / File: emd_11803.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 300 pix.
= 255. Å		0.85 Å/pix.
x 300 pix.
= 255. Å		0.85 Å/pix.
x 300 pix.
= 255. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.21
Minimum - Maximum-0.5021309 - 1.0164343
Average (Standard dev.)0.0049352786 (±0.038345706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 255.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.850.850.85
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z255.000255.000255.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.5021.0160.005

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Supplemental data

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Mask #1

Fileemd_11803_msk_1.map
Projections & Slices
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Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_11803_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11803_half_map_2.map
Projections & Slices
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Sample components

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Entire : Homodimeric complex of human potassium chloride transporter KCC1 ...

EntireName: Homodimeric complex of human potassium chloride transporter KCC1 in complex with ATP and magnesium
Components
  • Complex: Homodimeric complex of human potassium chloride transporter KCC1 in complex with ATP and magnesium
    • Protein or peptide: Solute carrier family 12 member 4
  • Protein or peptide: Solute carrier family 12 member 4
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Homodimeric complex of human potassium chloride transporter KCC1 ...

SupramoleculeName: Homodimeric complex of human potassium chloride transporter KCC1 in complex with ATP and magnesium
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: Solute carrier family 12 member 4

MacromoleculeName: Solute carrier family 12 member 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 119.610281 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLEGLSWVDY GERAELDDSD GHGNHRESSP FLSPLEASRG IDYYDRNLAL FEEELDIRPK VSSLLGKLVS YTNLTQGAKE HEEAESGEG TRRRAAEAPS MGTLMGVYLP CLQNIFGVIL FLRLTWMVGT AGVLQALLIV LICCCCTLLT AISMSAIATN G VVPAGGSY ...String:
MLEGLSWVDY GERAELDDSD GHGNHRESSP FLSPLEASRG IDYYDRNLAL FEEELDIRPK VSSLLGKLVS YTNLTQGAKE HEEAESGEG TRRRAAEAPS MGTLMGVYLP CLQNIFGVIL FLRLTWMVGT AGVLQALLIV LICCCCTLLT AISMSAIATN G VVPAGGSY FMISRSLGPE FGGAVGLCFY LGTTFAAAMY ILGAIEILLT YIAPPAAIFY PSGAHDTSNA TLNNMRVYGT IF LTFMTLV VFVGVKYVNK FASLFLACVI ISILSIYAGG IKSIFDPPVF PVCMLGNRTL SRDQFDICAK TAVVDNETVA TQL WSFFCH SPNLTTDSCD PYFMLNNVTE IPGIPGAAAG VLQENLWSAY LEKGDIVEKH GLPSADAPSL KESLPLYVVA DIAT SFTVL VGIFFPSVTG IMAGSNRSGD LRDAQKSIPV GTILAIITTS LVYFSSVVLF GACIEGVVLR DKYGDGVSRN LVVGT LAWP SPWVIVIGSF FSTCGAGLQS LTGAPRLLQA IAKDNIIPFL RVFGHGKVNG EPTWALLLTA LIAELGILIA SLDMVA PIL SMFFLMCYLF VNLACAVQTL LRTPNWRPRF KYYHWALSFL GMSLCLALMF VSSWYYALVA MLIAGMIYKY IEYQGAE KE WGDGIRGLSL SAARYALLRL EEGPPHTKNW RPQLLVLLKL DEDLHVKYPR LLTFASQLKA GKGLTIVGSV IQGSFLES Y GEAQAAEQTI KNMMEIEKVK GFCQVVVASK VREGLAHLIQ SCGLGGMRHN SVVLGWPYGW RQSEDPRAWK TFIDTVRCT TAAHLALLVP KNIAFYPSNH ERYLEGHIDV WWIVHDGGML MLLPFLLRQH KVWRKCRMRI FTVAQMDDNS IQMKKDLAVF LYHLRLEAE VEVVEMHNSD ISAYTYERTL MMEQRSQMLR QMRLTKTERE REAQLVKDRH SALRLESLYS DEEDESAVGA D KIQMTWTR DKYMTETWDP SHAPDNFREL VHIKPDQSNV RRMHTAVKLN EVIVTRSHDA RLVLLNMPGP PRNSEGDENY ME FLEVLTE GLERVLLVRG GGREVITIYS AENLYFQ

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Macromolecule #2: Solute carrier family 12 member 4

MacromoleculeName: Solute carrier family 12 member 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 119.69025 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLEGLSWVDY GERAELDDSD GHGNHRESSP FLSPLEASRG IDYYDRNLAL FEEELDIRPK VSSLLGKLVS YTNLTQGAKE HEEAESGEG TRRRAAEAPS MGTLMGVYLP CLQNIFGVIL FLRLTWMVGT AGVLQALLIV LICCCCTLLT AISMSAIATN G VVPAGGSY ...String:
MLEGLSWVDY GERAELDDSD GHGNHRESSP FLSPLEASRG IDYYDRNLAL FEEELDIRPK VSSLLGKLVS YTNLTQGAKE HEEAESGEG TRRRAAEAPS MGTLMGVYLP CLQNIFGVIL FLRLTWMVGT AGVLQALLIV LICCCCTLLT AISMSAIATN G VVPAGGSY FMISRSLGPE FGGAVGLCFY LGTTFAAAMY ILGAIEILLT YIAPPAAIFY PSGAHDTSNA TLNNMRVYGT IF LTFMTLV VFVGVKYVNK FASLFLACVI ISILSIYAGG IKSIFDPPVF PVCMLGNRTL SRDQFDICAK TAVVDNETVA TQL WSFFCH SPNLTTDSCD PYFMLNNVTE IPGIPGAAAG VLQENLWSAY LEKGDIVEKH GLPSADAPSL KESLPLYVVA DIAT SFTVL VGIFFPSVTG IMAGSNRSGD LRDAQKSIPV GTILAIITTS LVYFSSVVLF GACIEGVVLR DKYGDGVSRN LVVGT LAWP SPWVIVIGSF FSTCGAGLQS LTGAPRLLQA IAKDNIIPFL RVFGHGKVNG EPTWALLLTA LIAELGILIA SLDMVA PIL SMFFLMCYLF VNLACAVQTL LRTPNWRPRF KYYHWALSFL GMSLCLALMF VSSWYYALVA MLIAGMIYKY IEYQGAE KE WGDGIRGLSL SAARYALLRL EEGPPHTKNW RPQLLVLLKL DEDLHVKYPR LLTFASQLKA GKGLTIVGSV IQG(SEP) FLESY GEAQAAEQTI KNMMEIEKVK GFCQVVVASK VREGLAHLIQ SCGLGGMRHN SVVLGWPYGW RQSEDPRAWK TFIDT VRCT TAAHLALLVP KNIAFYPSNH ERYLEGHIDV WWIVHDGGML MLLPFLLRQH KVWRKCRMRI FTVAQMDDNS IQMKKD LAV FLYHLRLEAE VEVVEMHNSD ISAYTYERTL MMEQRSQMLR QMRLTKTERE REAQLVKDRH SALRLESLYS DEEDESA VG ADKIQMTWTR DKYMTETWDP SHAPDNFREL VHIKPDQSNV RRMHTAVKLN EVIVTRSHDA RLVLLNMPGP PRNSEGDE N YMEFLEVLTE GLERVLLVRG GGREVITIYS AENLYFQ

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 76330
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel from reference map/model fitted with Chimera, refined with Coot and then with Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7air:
Structure of Human Potassium Chloride Transporter KCC1 in NaCl (Subclass 2)

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