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- PDB-8qqi: E.coli DNA gyrase in complex with 217 bp substrate DNA and LEI-800 -

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Basic information

Entry
Database: PDB / ID: 8qqi
TitleE.coli DNA gyrase in complex with 217 bp substrate DNA and LEI-800
Components
  • (DNA gyrase subunit ...) x 2
  • Mu217 chain E
  • Mu217 chain F
KeywordsDNA BINDING PROTEIN / TOPOISOMERASE / GYRASE / ALLOSTERIC INHIBITOR / ANTIBIOTIC
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus / response to antibiotic ...DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex / DNA negative supercoiling activity / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / ATP-dependent activity, acting on DNA / DNA-templated DNA replication / chromosome / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / DNA binding / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase/topoisomerase IV, subunit A, C-terminal / : / DNA gyrase C-terminal domain, beta-propeller / DNA gyrase subunit B, TOPRIM domain ...: / GyrB, hook / DNA gyrase subunit B insert domain / DNA gyrase B subunit insert domain / DNA gyrase, subunit A / DNA gyrase/topoisomerase IV, subunit A, C-terminal repeat / DNA gyrase/topoisomerase IV, subunit A, C-terminal / : / DNA gyrase C-terminal domain, beta-propeller / DNA gyrase subunit B, TOPRIM domain / DNA gyrase, subunit B / DNA topoisomerase, type IIA, subunit B / DNA gyrase B subunit, C-terminal / DNA gyrase B subunit, carboxyl terminus / Topoisomerase (Topo) IIA-type catalytic domain profile. / DNA topoisomerase, type IIA, alpha-helical domain superfamily / DNA topoisomerase, type IIA, domain A / DNA topoisomerase, type IIA, domain A, alpha-beta / DNA gyrase/topoisomerase IV, subunit A / DNA Topoisomerase IV / DNA topoisomerase, type IIA, subunit B, domain 2 / DNA gyrase B / DNA topoisomerase, type IIA / DNA topoisomerase, type IIA, conserved site / DNA topoisomerase II signature. / TopoisomeraseII / DNA topoisomerase, type IIA, subunit B, C-terminal / Toprim domain / DNA topoisomerase, type IIA-like domain superfamily / Toprim domain profile. / TOPRIM domain / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA gyrase subunit A / DNA gyrase subunit B
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage Mu (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGhilarov, D. / Martin, N.I. / van der Stelt, M.
Funding support United Kingdom, European Union, Netherlands, 4items
OrganizationGrant numberCountry
Wellcome Trust221868/Z/20/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P012523/1 United Kingdom
European Research Council (ERC)725523European Union
Netherlands Organisation for Scientific Research (NWO)16444 Netherlands
CitationJournal: Nat Chem / Year: 2024
Title: Discovery of isoquinoline sulfonamides as allosteric gyrase inhibitors with activity against fluoroquinolone-resistant bacteria.
Authors: Alexander T Bakker / Ioli Kotsogianni / Mariana Avalos / Jeroen M Punt / Bing Liu / Diana Piermarini / Berend Gagestein / Cornelis J Slingerland / Le Zhang / Joost J Willemse / Leela B ...Authors: Alexander T Bakker / Ioli Kotsogianni / Mariana Avalos / Jeroen M Punt / Bing Liu / Diana Piermarini / Berend Gagestein / Cornelis J Slingerland / Le Zhang / Joost J Willemse / Leela B Ghimire / Richard J H B N van den Berg / Antonius P A Janssen / Tom H M Ottenhoff / Constant A A van Boeckel / Gilles P van Wezel / Dmitry Ghilarov / Nathaniel I Martin / Mario van der Stelt /
Abstract: Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of ...Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase-LEI-800-DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones.
History
DepositionOct 4, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA gyrase subunit A
B: DNA gyrase subunit B
C: DNA gyrase subunit A
D: DNA gyrase subunit B
E: Mu217 chain E
F: Mu217 chain F
hetero molecules


Theoretical massNumber of molelcules
Total (without water)313,84310
Polymers312,9066
Non-polymers9384
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA gyrase subunit ... , 2 types, 4 molecules ACBD

#1: Protein DNA gyrase subunit A


Mass: 58824.207 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MG1655 / Gene: gyrA / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star / References: UniProt: A0A3K0QBM9
#2: Protein DNA gyrase subunit B / Type IIA topoisomerase subunit GyrB


Mass: 89641.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MG1655
Gene: gyrB, acrB, cou, himB, hisU, nalC, parA, pcbA, b3699, JW5625
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Star
References: UniProt: P0AES6, DNA topoisomerase (ATP-hydrolysing)

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DNA chain , 2 types, 2 molecules EF

#3: DNA chain Mu217 chain E


Mass: 7943.173 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Mu (virus) / Production host: Escherichia coli (E. coli) / Strain (production host): NEB stable
#4: DNA chain Mu217 chain F


Mass: 8031.157 Da / Num. of mol.: 1 / Mutation: C18837G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Mu (virus) / Production host: Escherichia coli (E. coli) / Strain (production host): NEB Stable

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-LRL / ~{N}-[[(2~{S},5~{R})-5-(4-pyridin-3-ylphenyl)pyrrolidin-2-yl]methyl]isoquinoline-5-sulfonamide


Mass: 444.549 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C25H24N4O2S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Ternary complex of E.coli DNA gyrase with 217 bp linear dsDNA fragment and LEI-800
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MG1655
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: 25 mM Na-HEPES pH 8 KOAc 30 mM MgOAc 2.5 mM TCEP 0.5 mM CHAPSO 8 mM
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 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 defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 39.58 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU3.4image acquisition
4cryoSPARC4.2CTF correction
8PHENIXmodel refinement
10cryoSPARC4.2initial Euler assignment
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1810321
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139848 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316026
ELECTRON MICROSCOPYf_angle_d0.56221887
ELECTRON MICROSCOPYf_dihedral_angle_d12.7156250
ELECTRON MICROSCOPYf_chiral_restr0.0432448
ELECTRON MICROSCOPYf_plane_restr0.0052734

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