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- PDB-8p1i: Single particle cryo-EM co-structure of Klebsiella pneumoniae Acr... -

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Basic information

Entry
Database: PDB / ID: 8p1i
TitleSingle particle cryo-EM co-structure of Klebsiella pneumoniae AcrB with the BDM91288 efflux pump inhibitor at 2.97 Angstrom resolution
ComponentsEfflux pump membrane transporter
KeywordsTRANSPORT PROTEIN / Efflux Pump Inhibitor / Transmembrane Binding / Antibiotic Resistance
Function / homologyChem-PG8 / 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline / Chem-WR6 / :
Function and homology information
Biological speciesKlebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsBoernsen, C. / Mueller, R.T. / Pos, K.M. / Frangakis, A.S.
Funding support Germany, France, 2items
OrganizationGrant numberCountry
German Federal Ministry for Education and ResearchBMBF- 16GW0236K Germany
Agence Nationale de la Recherche (ANR)ANR-19-AMRB-0007 France
CitationJournal: EMBO Mol Med / Year: 2024
Title: Pyridylpiperazine efflux pump inhibitor boosts in vivo antibiotic efficacy against K. pneumoniae.
Authors: Anais Vieira Da Cruz / Juan-Carlos Jiménez-Castellanos / Clara Börnsen / Laurye Van Maele / Nina Compagne / Elizabeth Pradel / Reinke T Müller / Virginie Meurillon / Daphnée Soulard / ...Authors: Anais Vieira Da Cruz / Juan-Carlos Jiménez-Castellanos / Clara Börnsen / Laurye Van Maele / Nina Compagne / Elizabeth Pradel / Reinke T Müller / Virginie Meurillon / Daphnée Soulard / Catherine Piveteau / Alexandre Biela / Julie Dumont / Florence Leroux / Benoit Deprez / Nicolas Willand / Klaas M Pos / Achilleas S Frangakis / Ruben C Hartkoorn / Marion Flipo /
Abstract: Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump ...Antimicrobial resistance is a global problem, rendering conventional treatments less effective and requiring innovative strategies to combat this growing threat. The tripartite AcrAB-TolC efflux pump is the dominant constitutive system by which Enterobacterales like Escherichia coli and Klebsiella pneumoniae extrude antibiotics. Here, we describe the medicinal chemistry development and drug-like properties of BDM91288, a pyridylpiperazine-based AcrB efflux pump inhibitor. In vitro evaluation of BDM91288 confirmed it to potentiate the activity of a panel of antibiotics against K. pneumoniae as well as revert clinically relevant antibiotic resistance mediated by acrAB-tolC overexpression. Using cryo-EM, BDM91288 binding to the transmembrane region of K. pneumoniae AcrB was confirmed, further validating the mechanism of action of this inhibitor. Finally, proof of concept studies demonstrated that oral administration of BDM91288 significantly potentiated the in vivo efficacy of levofloxacin treatment in a murine model of K. pneumoniae lung infection.
History
DepositionMay 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Efflux pump membrane transporter
B: Efflux pump membrane transporter
C: Efflux pump membrane transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)345,5507
Polymers343,4703
Non-polymers2,0804
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Efflux pump membrane transporter


Mass: 114489.945 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
Strain: FDAARGOS_775 / Gene: UUU_08720 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): C43(DE3)deltaacrAB / References: UniProt: J2LW81
#2: Chemical ChemComp-WQW / 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline


Mass: 333.859 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H24ClN5 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-WR6 / [(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-3-undecanoyloxy-propan-2-yl] tricosanoate


Mass: 751.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H79O10P
#4: Chemical ChemComp-PG8 / 1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)


Mass: 497.537 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H42O10P
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: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inhibitor
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Klebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
Strain: FDAARGOS_775
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43(DE3)deltaacrAB
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMsodium chlorideNaClSodium chloride1
30.02 %DDM1
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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: 130000 X / Calibrated magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8487
EM imaging opticsEnergyfilter name: GIF Quantum SE / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4CTF correction
12cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 380181
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 168689 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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