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- PDB-6sgs: Cryo-EM structure of Escherichia coli AcrBZ and DARPin in Saposin... -

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Basic information

Entry
Database: PDB / ID: 6sgs
TitleCryo-EM structure of Escherichia coli AcrBZ and DARPin in Saposin A-nanodisc
Components
  • DARPin
  • Multidrug efflux pump accessory protein AcrZ
  • Multidrug efflux pump subunit AcrB
KeywordsMEMBRANE PROTEIN / RND transporter / efflux pump / drug transport / antibiotic resistance / lipid nanodisc
Function / homology
Function and homology information


xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / outer membrane-bounded periplasmic space / membrane => GO:0016020 / response to antibiotic / membrane ...xenobiotic detoxification by transmembrane export across the cell outer membrane / efflux pump complex / periplasmic side of plasma membrane / xenobiotic detoxification by transmembrane export across the plasma membrane / xenobiotic transmembrane transporter activity / efflux transmembrane transporter activity / outer membrane-bounded periplasmic space / membrane => GO:0016020 / response to antibiotic / membrane / identical protein binding / plasma membrane
Similarity search - Function
Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump-associated protein AcrZ / Hydrophobe/amphiphile efflux-1 HAE1 / Acriflavin resistance protein / Multidrug efflux transporter AcrB TolC docking domain, DN/DC subdomains / AcrB/AcrD/AcrF family / Ankyrin repeat-containing domain / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Mainly Alpha
Similarity search - Domain/homology
Multidrug efflux pump accessory protein AcrZ / Multidrug efflux pump subunit AcrB
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria)
synthetic construct (others)
Shigella boydii 965-58 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSzewczak-Harris, A. / Du, D. / Newman, C. / Neuberger, A. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
European Research Council742210 United Kingdom
CitationJournal: Structure / Year: 2020
Title: Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.
Authors: Dijun Du / Arthur Neuberger / Mona Wu Orr / Catherine E Newman / Pin-Chia Hsu / Firdaus Samsudin / Andrzej Szewczak-Harris / Leana M Ramos / Mekdes Debela / Syma Khalid / Gisela Storz / Ben F Luisi /
Abstract: The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic ...The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.
History
DepositionAug 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 10, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: Multidrug efflux pump subunit AcrB
B: Multidrug efflux pump subunit AcrB
C: Multidrug efflux pump subunit AcrB
D: DARPin
E: DARPin
F: Multidrug efflux pump accessory protein AcrZ
G: Multidrug efflux pump accessory protein AcrZ
H: Multidrug efflux pump accessory protein AcrZ


Theoretical massNumber of molelcules
Total (without water)393,5448
Polymers393,5448
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31010 Å2
ΔGint-194 kcal/mol
Surface area127720 Å2
MethodPISA

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Components

#1: Protein Multidrug efflux pump subunit AcrB / AcrAB-TolC multidrug efflux pump subunit AcrB / Acridine resistance protein B


Mass: 113665.180 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: acrB, acrE, b0462, JW0451
Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria)
References: UniProt: P31224
#2: Protein DARPin /


Mass: 18317.566 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria)
#3: Protein/peptide Multidrug efflux pump accessory protein AcrZ / AcrAB-TolC multidrug efflux pump accessory protein AcrZ / Acridine resistance protein Z


Mass: 5304.423 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella boydii 965-58 (bacteria) / Gene: acrZ, SB96558_0924
Production host: Escherichia coli str. K-12 substr. MG1655 (bacteria)
References: UniProt: I6DQK7

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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

Component
IDNameTypeEntity IDParent-IDSource
1Ternary complex of AcrBZ and DARPin in Saposin A-nanodiscCOMPLEXall0MULTIPLE SOURCES
2Multidrug efflux pump subunit AcrBCOMPLEX#11RECOMBINANT
3DARPinCOMPLEX#21RECOMBINANT
4Multidrug efflux pump accessory protein AcrZCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23synthetic construct (others)32630
34Shigella boydii 965-58 (bacteria)766138
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli str. K-12 substr. MG1655 (bacteria)511145
23Escherichia coli str. K-12 substr. MG1655 (bacteria)511145
34Escherichia coli str. K-12 substr. MG1655 (bacteria)511145
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(HOCH2)3CNH21
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
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: 100 % / Chamber temperature: 277.15 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 FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 3400 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 26.25 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1356
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4Gctf1.08CTF correction
11RELION3classification
12RELION33D reconstruction
13PHENIX1.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94507 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00354659
ELECTRON MICROSCOPYf_angle_d0.51699179
ELECTRON MICROSCOPYf_dihedral_angle_d7.46923433
ELECTRON MICROSCOPYf_chiral_restr0.0394379
ELECTRON MICROSCOPYf_plane_restr0.0038123

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