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- PDB-8by2: Structure of the K+/H+ exchanger KefC with GSH. -

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Basic information

Entry
Database: PDB / ID: 8by2
TitleStructure of the K+/H+ exchanger KefC with GSH.
ComponentsGlutathione-regulated potassium-efflux system protein KefC
KeywordsMEMBRANE PROTEIN / potassium proton exchanger / KefC / Transporter / CPA / GSH
Function / homology
Function and homology information


glutathione-regulated potassium exporter activity / response to methylglyoxal / antiporter activity / toxic substance binding / proton transmembrane transport / enzyme binding / plasma membrane
Similarity search - Function
Glutathione-regulated potassium-efflux system protein KefC / K+/H+ exchanger / Potassium uptake protein TrkA / Sodium/solute symporter superfamily / Cation/H+ exchanger / Sodium/hydrogen exchanger family / Regulator of K+ conductance, N-terminal / TrkA-N domain / RCK N-terminal domain profile. / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / GLUTATHIONE / : / Chem-PGW / Glutathione-regulated potassium-efflux system protein KefC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsGulati, A. / Drew, D.
Funding supportEuropean Union, Sweden, 2items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-CoG-820187European Union
Swedish Research Council Sweden
CitationJournal: Nat Commun / Year: 2024
Title: Structure and mechanism of the K/H exchanger KefC.
Authors: Ashutosh Gulati / Surabhi Kokane / Annemarie Perez-Boerema / Claudia Alleva / Pascal F Meier / Rei Matsuoka / David Drew /
Abstract: Intracellular potassium (K) homeostasis is fundamental to cell viability. In addition to channels, K levels are maintained by various ion transporters. One major family is the proton-driven K efflux ...Intracellular potassium (K) homeostasis is fundamental to cell viability. In addition to channels, K levels are maintained by various ion transporters. One major family is the proton-driven K efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na/H antiporter NapA. By structural assignment of a coordinated K ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K ion. KefC harbors C-terminal regulator of K conductance (RCK) domains, as present in some bacterial K-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K-ion-channels.
History
DepositionDec 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Structure summary / Category: citation / struct / Item: _citation.journal_abbrev / _struct.title
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutathione-regulated potassium-efflux system protein KefC
B: Glutathione-regulated potassium-efflux system protein KefC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,25210
Polymers122,3662
Non-polymers2,8858
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14250 Å2
ΔGint-95 kcal/mol
Surface area47970 Å2
MethodPISA

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Components

#1: Protein Glutathione-regulated potassium-efflux system protein KefC / K(+)/H(+) antiporter


Mass: 61183.223 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: kefC, kefC_3, kefC_4, A9X72_21045, ACU57_16285, AT845_001330, BHS81_26455, BJJ90_21970, BLM69_001595, BMT91_01970, BN17_44701, BON64_08355, BON68_06240, BON72_08730, BON76_14820, BON94_26490, ...Gene: kefC, kefC_3, kefC_4, A9X72_21045, ACU57_16285, AT845_001330, BHS81_26455, BJJ90_21970, BLM69_001595, BMT91_01970, BN17_44701, BON64_08355, BON68_06240, BON72_08730, BON76_14820, BON94_26490, BON97_15340, BRV02_001703, BTQ06_18350, BvCmsF30A_01124, BvCmsHHP056_04946, BvCmsKKP061_01751, BvCmsKSNP073_00849, BVL39_02825, C0P57_001700, C1Q91_003817, CCS08_23225, CIG67_13365, CR538_21280, CV83915_01528, CWS33_08975, D3Y67_20250, D9H94_05730, DAH19_13555, DAH22_04310, DAH27_12485, DAH28_04320, DAH29_14050, DAH30_11255, DAH32_09665, DAH37_05870, DAH41_08355, DEN89_19705, DEN90_18045, DEN96_05545, DEN97_05070, DEN98_05075, DEN99_02470, DEO00_04545, DEO12_14740, DEO14_11885, DEO19_04855, DIV22_05610, DRW19_05570, DS732_05130, DTL43_06565, DXT69_02135, DXT71_01260, E2121_11235, E2122_03850, E2127_05310, E2128_11280, E2129_03465, E2131_02880, E2132_05315, E2135_03750, E5P26_16110, E5P27_07520, E5P28_15370, E5P29_09025, E5P30_07860, E5P39_14645, E5P41_09555, E5P42_00460, E5P43_10360, E5P44_10710, E5P45_09675, E5P46_12140, E5P47_12165, E5P48_11095, E5P49_12390, E5P50_01615, E5P51_19605, E5S35_00760, E5S38_08180, E5S42_10720, E5S43_02390, E5S45_05455, E5S52_10125, E6D34_01595, EAI46_08450, EBP16_09705, EC95NR1_04226, EHD79_22595, EKI52_11150, EL79_3823, EL80_3769, ELT21_05100, ELV08_16095, ELY39_00725, ERS139208_01108, F0L67_09735, FDM60_06480, FJQ40_06620, FPJ29_04385, G3V95_03045, G4A38_11120, G4A47_11350, GAI66_03965, GF699_05360, GNZ05_10415, GOP25_07850, GP944_03730, GP965_24280, GQM04_06640, GRW24_15515, GUC01_17280, HNC36_14075, HV209_00940, J5U05_003215, JFD_01135, JNP96_04415, NCTC10865_05215, NCTC8960_01687, NCTC9036_04147, SAMEA3753106_01628, WR15_01725
Production host: Escherichia coli (E. coli) / References: UniProt: J7Q5Y6
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#4: Chemical ChemComp-GSH / GLUTATHIONE


Mass: 307.323 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N3O6S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H77O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
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

ComponentName: Kefc protein dimer with GSH / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 64.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 260115 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 82.62 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028700
ELECTRON MICROSCOPYf_angle_d0.49411755
ELECTRON MICROSCOPYf_dihedral_angle_d9.831280
ELECTRON MICROSCOPYf_chiral_restr0.0341396
ELECTRON MICROSCOPYf_plane_restr0.0041459

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