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Yorodumi- PDB-6y5v: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T94... -
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-Basic information
Entry | Database: PDB / ID: 6y5v | |||||||||
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Title | Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl | |||||||||
Components | Solute carrier family 12 member 6 | |||||||||
Keywords | MEMBRANE PROTEIN / Dimer / transporter / potassium chloride / KCC3 / SLC12A6 / APC / Structural Genomics / Structural Genomics Consortium / SGC | |||||||||
Function / homology | Function and homology information Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / ammonium import across plasma membrane / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / ammonium channel activity / cellular hypotonic salinity response / potassium ion homeostasis ...Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / ammonium import across plasma membrane / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / ammonium channel activity / cellular hypotonic salinity response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / cellular response to glucose stimulus / chemical synaptic transmission / basolateral plasma membrane / angiogenesis / axon / synapse / protein kinase binding / membrane / metal ion binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.08 Å | |||||||||
Authors | Chi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, E.M. / Chalk, R. ...Chi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, E.M. / Chalk, R. / Moreau, C. / Snee, M. / Bohstedt, T. / Singh, N.K. / Abrusci, P. / Arrowsmith, C.H. / Bountra, C. / Edwards, A.M. / Marsden, B.D. / Burgess-Brown, N.A. / DiMaio, F. / Duerr, K.L. | |||||||||
Funding support | United Kingdom, 1items
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Citation | Journal: 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 |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
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PDBx/mmCIF format | 6y5v.cif.gz | 316.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6y5v.ent.gz | 249.2 KB | Display | PDB format |
PDBx/mmJSON format | 6y5v.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6y5v_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 6y5v_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 6y5v_validation.xml.gz | 60.9 KB | Display | |
Data in CIF | 6y5v_validation.cif.gz | 89.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/y5/6y5v ftp://data.pdbj.org/pub/pdb/validation_reports/y5/6y5v | HTTPS FTP |
-Related structure data
Related structure data | 10704MC 7ainC 7aioC 7aipC 7aiqC 7airC 7ngbC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 122344.422 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A6, KCC3 / Production host: Homo sapiens (human) / References: UniProt: Q9UHW9 #2: Polysaccharide | Source method: isolated from a genetically manipulated source #3: Sugar | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Structure of Human Potassium Chloride Transporter KCC3b (S45D/T940D/T997D) in KCl Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.24 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Homo sapiens (human) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software |
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EM software | Name: EPU / Category: image acquisition | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 293233 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 65.95 Å2 | ||||||||||||||||||||||||
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