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- PDB-7aip: Structure of Human Potassium Chloride Transporter KCC1 in NaCl (R... -

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Basic information

Entry
Database: PDB / ID: 7aip
TitleStructure of Human Potassium Chloride Transporter KCC1 in NaCl (Reference Map)
ComponentsSolute carrier family 12 member 4
KeywordsTRANSPORT PROTEIN / CCC / transporter / human membrane protein / homodimer / nucleotide-binding / KCC1 / KCC / potassium-chloride coupled transporter / Structural Genomics / Structural Genomics Consortium / SGC
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
ADENOSINE-5'-TRIPHOSPHATE / Solute carrier family 12 member 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsEbenhoch, R. / Chi, G. / Man, H. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, E.M. / Chalk, R. / Moreau, C. / Snee, M. ...Ebenhoch, R. / Chi, G. / Man, H. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, E.M. / Chalk, R. / Moreau, C. / Snee, M. / Bohstedt, T. / Liko, I. / Tehan, B.G. / Almeida, F.G. / Elkins, J. / Singh, N.K. / Abrusci, P. / Arrowsmith, C.H. / Tang, H. / Robinson, C.V. / Bountra, C. / Edwards, A.M. / Marsden, B.D. / Burgess-Brown, N.A. / Duerr, K.L. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 1items
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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 9, 2021Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Nov 20, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-11801
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Solute carrier family 12 member 4
B: Solute carrier family 12 member 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,62510
Polymers239,5402
Non-polymers3,0858
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10730 Å2
ΔGint-42 kcal/mol
Surface area74760 Å2
MethodPISA

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Solute carrier family 12 member 4 / Electroneutral potassium-chloride cotransporter 1 / Erythroid K-Cl cotransporter 1 / hKCC1


Mass: 119770.234 Da / Num. of mol.: 2 / Mutation: delta19
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A4, KCC1 / Production host: Homo sapiens (human) / References: UniProt: Q9UP95

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Sugars , 2 types, 4 molecules

#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 3 types, 8 molecules

#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Homodimeric complex of human potassium chloride transporter KCC1 in complex with ATP and magnesium
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
7UCSF Chimeramodel fitting
9Cootmodel refinement
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 818813 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: Model fitting with UCSF Chimera, sequence replacement with Chainsaw, manual refinement with Coot, and then real-space automated refinement with Phenix was done.
Atomic model buildingPDB-ID: 6Y5V

6y5v


Accession code: 6Y5V / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006413841
ELECTRON MICROSCOPYf_angle_d0.782518866
ELECTRON MICROSCOPYf_chiral_restr0.04822231
ELECTRON MICROSCOPYf_plane_restr0.00462318
ELECTRON MICROSCOPYf_dihedral_angle_d17.00684896

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