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- PDB-8gcc: T. cruzi topoisomerase II alpha bound to dsDNA and the covalent i... -

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Basic information

Entry
Database: PDB / ID: 8gcc
TitleT. cruzi topoisomerase II alpha bound to dsDNA and the covalent inhibitor CT1
Components
  • DNA (28-MER)
  • DNA topoisomerase 2
KeywordsISOMERASE / Topoisomerase / DNA binding protein / Topoisomerase inhibitor
Function / homology
Function and homology information


DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / DNA topological change / DNA binding / ATP binding / metal ion binding
Similarity search - Function
DNA topoisomerase 2, TOPRIM domain / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / 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 2, TOPRIM domain / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / DNA topoisomerase II, eukaryotic-type / : / 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 / DNA topoisomerase, type IIA-like domain superfamily / Toprim domain profile. / TOPRIM domain / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase/HSP90-like ATPase / 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
Chem-YWX / DNA / DNA (> 10) / DNA topoisomerase 2
Similarity search - Component
Biological speciesTrypanosoma cruzi strain CL Brener (eukaryote)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsSchenk, A. / Deniston, C. / Noeske, J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust219639/Z/19/Z United Kingdom
Wellcome Trust108517/Z/15/Z United Kingdom
CitationJournal: Science / Year: 2023
Title: Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.
Authors: Srinivasa P S Rao / Matthew K Gould / Jonas Noeske / Manuel Saldivia / Rajiv S Jumani / Pearly S Ng / Olivier René / Yen-Liang Chen / Marcel Kaiser / Ryan Ritchie / Amanda Fortes Francisco ...Authors: Srinivasa P S Rao / Matthew K Gould / Jonas Noeske / Manuel Saldivia / Rajiv S Jumani / Pearly S Ng / Olivier René / Yen-Liang Chen / Marcel Kaiser / Ryan Ritchie / Amanda Fortes Francisco / Nila Johnson / Debjani Patra / Harry Cheung / Colin Deniston / Andreas D Schenk / Wilian A Cortopassi / Remo S Schmidt / Natalie Wiedemar / Bryanna Thomas / Rima Palkar / Nahdiyah A Ghafar / Vanessa Manoharan / Catherine Luu / Jonathan E Gable / Kah Fei Wan / Elmarie Myburgh / Jeremy C Mottram / Whitney Barnes / John Walker / Charles Wartchow / Natasha Aziz / Colin Osborne / Juergen Wagner / Christopher Sarko / John M Kelly / Ujjini H Manjunatha / Pascal Mäser / Jan Jiricek / Suresh B Lakshminarayana / Michael P Barrett / Thierry T Diagana /
Abstract: Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are ...Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.
History
DepositionMar 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 12, 2023Provider: 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 topoisomerase 2
B: DNA topoisomerase 2
C: DNA (28-MER)
D: DNA (28-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,6516
Polymers195,8544
Non-polymers7972
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA topoisomerase 2


Mass: 89322.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi strain CL Brener (eukaryote)
Gene: Tc00.1047053508699.10 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q4DE53
#2: DNA chain DNA (28-MER)


Mass: 8604.566 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: Chemical ChemComp-YWX / 2-{3-[(Z)-iminomethyl]-1H-1,2,4-triazol-1-yl}-1-{(3M)-3-[2-(trifluoromethyl)phenyl]-6H-pyrrolo[3,4-b]pyridin-6-yl}ethan-1-one / inhibitor CT1, bound form


Mass: 398.341 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H13F3N6O / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Topoisomerase II alpha bound to dsDNA and inhibitor CT1COMPLEX#1-#20RECOMBINANT
2Topoisomerase II alphaCOMPLEX#11RECOMBINANT
3dsDNACOMPLEX#22SYNTHETICDNA sequence: GG GAT AAC AAT GAG CTC ATT GTT ATC CC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Trypanosoma cruzi (eukaryote)5693
32Trypanosoma cruzi (eukaryote)5693
43Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
43Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
10.02 MHEPES1
20.1 Mpotassium chlorideKCl1
30.003 Mmagnesium chlorideMgCl21
40.001 MTCEP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
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 FIELD / Nominal magnification: 75000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1

IDAverage exposure time (sec.)Num. of real imagesDetails
12.83014Micrographs collected at 30 degrees tilt using a Falcon 4i camera.
22.982220Micrographs collected at 30 degrees tilt using a Falcon 4 camera.
Image scans
WidthHeightIDImage recording-IDEntry-ID
40964096118GCC
40964096228GCC

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPU3image acquisition
4cryoSPARC4CTF correction
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
Image processingDetails: Untilted and tilted data merged
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1659398
Details: 955991 particles for tilted data 703407 particles for untilted data
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179787 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412063
ELECTRON MICROSCOPYf_angle_d0.49816584
ELECTRON MICROSCOPYf_dihedral_angle_d17.0032079
ELECTRON MICROSCOPYf_chiral_restr0.0391866
ELECTRON MICROSCOPYf_plane_restr0.0031997

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