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- EMDB-17350: Single particle cryo-EM co-structure of Klebsiella pneumoniae Acr... -

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Basic information

Entry
Database: EMDB / ID: EMD-17350
TitleSingle particle cryo-EM co-structure of Klebsiella pneumoniae AcrB with the BDM91288 efflux pump inhibitor at 2.97 Angstrom resolution
Map data
Sample
  • Complex: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inhibitor
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline
  • Ligand: [(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-3-undecanoyloxy-propan-2-yl] tricosanoate
  • Ligand: 1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)
KeywordsEfflux Pump Inhibitor / Transmembrane Binding / Antibiotic Resistance / TRANSPORT PROTEIN
Function / homology:
Function and homology information
Biological speciesKlebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsBoernsen C / Mueller RT / Pos KM / Frangakis AS
Funding support Germany, France, 2 items
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, 2023-
Header (metadata) releaseJan 17, 2024-
Map releaseJan 17, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17350.png

Downloads & links

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Map

FileDownload / File: emd_17350.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 380 pix.
= 318.06 Å		0.84 Å/pix.
x 380 pix.
= 318.06 Å		0.84 Å/pix.
x 380 pix.
= 318.06 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.837 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.571
Minimum - Maximum-2.990159 - 3.9581587
Average (Standard dev.)0.0036977837 (±0.13446926)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 318.06 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17350_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_17350_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_17350_half_map_2.map
Projections & Slices
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Sample components

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Entire : Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inh...

EntireName: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inhibitor
Components
  • Complex: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inhibitor
    • Protein or peptide: Efflux pump membrane transporter
  • Ligand: 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline
  • Ligand: [(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-3-undecanoyloxy-propan-2-yl] tricosanoate
  • Ligand: 1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)

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Supramolecule #1: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inh...

SupramoleculeName: Trimeric Klebsiella pneumoniae AcrB with BDM91288 efflux pump inhibitor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Klebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
Strain: FDAARGOS_775

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Macromolecule #1: Efflux pump membrane transporter

MacromoleculeName: Efflux pump membrane transporter / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae subsp. pneumoniae DSM 30104 = JCM 1662 = NBRC 14940 (bacteria)
Strain: FDAARGOS_775
Molecular weightTheoretical: 114.489945 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPNFFIDRPI FAWVIAIIIM LAGGLSILKL PVAQYPTIAP PAISITAMYP GADAETVQNT VTQVIEQNMN GIDHLMYMSS NGDSTGTAT ITLTFESGTD PDIAQVQVQN KLALATPLLP QEVQQQGISV EKASSSFLMV VGVINTNGTM NQDDISDYVA A NMKDPISR ...String:
MPNFFIDRPI FAWVIAIIIM LAGGLSILKL PVAQYPTIAP PAISITAMYP GADAETVQNT VTQVIEQNMN GIDHLMYMSS NGDSTGTAT ITLTFESGTD PDIAQVQVQN KLALATPLLP QEVQQQGISV EKASSSFLMV VGVINTNGTM NQDDISDYVA A NMKDPISR TSGVGDVQLF GSQYAMRIWM DPNKLNNFQL TPVDVISALK AQNAQVAAGQ LGGTPPVKGQ QLNASIIAQT RL TNTEEFG NILLKVNQDG SQVRLRDVAK IELGGESYDV VAKFNGQPAS GLGIKLATGA NALDTANAIR AELAKMEPFF PSG MKIVYP YDTTPFVKIS IHEVVKTLVE AIILVFLVMY LFLQNFRATL IPTIAVPVVL LGTFAVLAAF GFSINTLTMF GMVL AIGLL VDDAIVVVEN VERVMAEEGL PPKEATRKSM GQIQGALVGI AMVLSAVFIP MAFFGGSTGA IYRQFSITIV SAMAL SVLV ALILTPALCA TMLKPIQKGS HGATTGFFGW FNRMFDKSTH HYTDSVGNIL RSTGRYLVLY LIIVVGMAWL FVRLPS SFL PDEDQGVFLS MAQLPAGATQ ERTQKVLDEM TNYYLTKEKD NVESVFAVNG FGFAGRGQNT GIAFVSLKDW SQRPGEE NK VEAITARAMG YFSQIKDAMV FAFNLPAIVE LGTATGFDFE LIDQGGLGHE KLTQARNQLF GMVAQHPDVL TGVRPNGL E DTPQFKIDID QEKAQALGVS ISDINTTLGA AWGGSYVNDF IDRGRVKKVY IMSEAKYRML PEDIGKWYVR GSDGQMVPF SAFSTSRWEY GSPRLERYNG LPSLEILGQA APGKSTGEAM ALMEELAGKL PSGIGYDWTG MSYQERLSGN QAPALYAISL IVVFLCLAA LYESWSIPFS VMLVVPLGVV GALLAATFRG LTNDVYFQVG LLTTIGLSAK NAILIVEFAK DLMEKEGKGL I EATLEAVR MRLRPILMTS LAFILGVMPL VISSGAGSGA QNAVGTGVMG GMVTATILAI FFVPVFFVVV RRRFSKKTED IE HSHQVEH HLEHHHHHH

UniProtKB: UNIPROTKB: J2LW81

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Macromolecule #2: 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline

MacromoleculeName: 3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline / type: ligand / ID: 2 / Number of copies: 1 / Formula: WQW
Molecular weightTheoretical: 333.859 Da
Chemical component information

ChemComp-WQW:
3-chloranyl-2,6-di(piperazin-4-ium-1-yl)quinoline

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Macromolecule #3: [(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphory...

MacromoleculeName: [(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-3-undecanoyloxy-propan-2-yl] tricosanoate
type: ligand / ID: 3 / Number of copies: 1 / Formula: WR6
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-WR6:
[(2~{R})-1-[[(2~{R})-2,3-bis(oxidanyl)propoxy]-oxidanyl-phosphoryl]oxy-3-undecanoyloxy-propan-2-yl] tricosanoate

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Macromolecule #4: 1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)

MacromoleculeName: 1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL) / type: ligand / ID: 4 / Number of copies: 2 / Formula: PG8
Molecular weightTheoretical: 497.537 Da
Chemical component information

ChemComp-PG8:
1,2-DIOCTANOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMsodium chlorideNaCl
0.02 %DDM
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum SE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8487 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 380181
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 168689
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8p1i:
Single particle cryo-EM co-structure of Klebsiella pneumoniae AcrB with the BDM91288 efflux pump inhibitor at 2.97 Angstrom resolution

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