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- PDB-8chb: Inward-facing conformation of the ABC transporter BmrA C436S/A582... -

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Basic information

Entry
Database: PDB / ID: 8chb
TitleInward-facing conformation of the ABC transporter BmrA C436S/A582C cross-linked mutant
ComponentsMultidrug resistance ABC transporter ATP-binding/permease protein BmrA
KeywordsMEMBRANE PROTEIN / ABC transporter / drug efflux pump / homodimer / antibiotic resistance
Function / homology
Function and homology information


ATPase-coupled lipid transmembrane transporter activity / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / ABC-type transporter activity / transmembrane transport / response to antibiotic / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsDi Cesare, M. / Kaplan, E. / Hanssen, E. / Valimehr, S. / Orelle, C. / Jault, J.M.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0023 France
CitationJournal: J Biol Chem / Year: 2024
Title: The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.
Authors: Margot Di Cesare / Elise Kaplan / Julia Rendon / Guillaume Gerbaud / Sepideh Valimehr / Alexia Gobet / Thu-Anh Thi Ngo / Vincent Chaptal / Pierre Falson / Marlène Martinho / Pierre Dorlet / ...Authors: Margot Di Cesare / Elise Kaplan / Julia Rendon / Guillaume Gerbaud / Sepideh Valimehr / Alexia Gobet / Thu-Anh Thi Ngo / Vincent Chaptal / Pierre Falson / Marlène Martinho / Pierre Dorlet / Eric Hanssen / Jean-Michel Jault / Cédric Orelle /
Abstract: ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug ...ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.
History
DepositionFeb 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2023Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 24, 2024Group: Database references / Category: citation / citation_author / Item: _citation.journal_volume / _citation_author.name
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA
B: Multidrug resistance ABC transporter ATP-binding/permease protein BmrA


Theoretical massNumber of molelcules
Total (without water)132,6822
Polymers132,6822
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Multidrug resistance ABC transporter ATP-binding/permease protein BmrA


Mass: 66340.836 Da / Num. of mol.: 2 / Mutation: C436S, A582C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: bmrA, yvcC, BSU34820 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: O06967, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
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 ATP-binding Cassette Transporter BmrA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41
Buffer solutionpH: 8 / Details: pH adjustment with sodium hydroxide
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acidC8H18N2O4S1
250 mMSodium ChlorideNaCl1
30.035 %n-Dodecyl-B-D-maltopyranosideC24H46O111
40.03 %Sodium CholateC24H39NaO51
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295.15 K

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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: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.8 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13937
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selectionblob picker was used to select particle images
2EPUimage acquisition
4cryoSPARC3.3.1CTF correctionPatch CTF estimation was used for the
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2057656
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 343396 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 90.1 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058674
ELECTRON MICROSCOPYf_angle_d0.61411738
ELECTRON MICROSCOPYf_dihedral_angle_d5.1391182
ELECTRON MICROSCOPYf_chiral_restr0.0381424
ELECTRON MICROSCOPYf_plane_restr0.0071468

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