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- PDB-9emb: Structure of KefC Asp156Asn variant -

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Basic information

Entry
Database: PDB / ID: 9emb
TitleStructure of KefC Asp156Asn variant
ComponentsGlutathione-regulated potassium-efflux system protein KefC
KeywordsTRANSPORT PROTEIN / KefC / Potassium / GSH / Mutant
Function / homology
Function and homology information


glutathione-regulated potassium exporter activity / response to methylglyoxal / potassium:proton antiporter complex / intracellular pH elevation / regulation of pH / antiporter activity / toxic substance binding / proton transmembrane transport / regulation of intracellular pH / potassium ion transport ...glutathione-regulated potassium exporter activity / response to methylglyoxal / potassium:proton antiporter complex / intracellular pH elevation / regulation of pH / antiporter activity / toxic substance binding / proton transmembrane transport / regulation of intracellular pH / potassium ion transport / response to hydrogen peroxide / response to toxic substance / nucleotide binding / enzyme binding / protein homodimerization activity / membrane / plasma membrane
Similarity search - Function
Glutathione-regulated potassium-efflux system protein KefC / K+/H+ exchanger / Potassium uptake protein TrkA / Sodium/solute symporter superfamily / Regulator of K+ conductance, N-terminal / TrkA-N domain / Cation/H+ exchanger / Sodium/hydrogen exchanger, transmembrane / RCK N-terminal domain profile. / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / 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: 2.98 Å
AuthorsGulati, A. / Kokane, S. / Drew, D.
Funding support Sweden, European Union, 2items
OrganizationGrant numberCountry
Swedish Research Council Sweden
European Research Council (ERC)European Union
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
DepositionMar 7, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details / struct_ref / struct_ref_seq / struct_ref_seq_dif
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 / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)124,5576
Polymers122,3642
Non-polymers2,1924
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12810 Å2
ΔGint-93 kcal/mol
Surface area48710 Å2
MethodPISA

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Components

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


Mass: 61182.238 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: kefC, trkC, b0047, JW0046 / Production host: Escherichia coli (E. coli) / References: UniProt: P03819
#2: 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
#3: 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
Has protein modificationN

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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: Homodimer of KefC Asp156Asn variant / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
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
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 / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 54.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 327001 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.78 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00248653
ELECTRON MICROSCOPYf_angle_d0.559611695
ELECTRON MICROSCOPYf_chiral_restr0.03441391
ELECTRON MICROSCOPYf_plane_restr0.00381449
ELECTRON MICROSCOPYf_dihedral_angle_d8.45211257

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