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- EMDB-34022: Cryo-EM structure of human topoisomerase II beta in complex with ... -

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Entry
Database: EMDB / ID: EMD-34022
TitleCryo-EM structure of human topoisomerase II beta in complex with DNA and etoposide
Map data
Sample
  • Complex: Ternary complex of human topoisomerase II beta with DNA and etoposide
    • Complex: human topoisomerase II beta
      • Protein or peptide: DNA topoisomerase 2-beta
    • Complex: DNA
      • DNA: 50-mer DNA
      • DNA: 50-mer DNA
  • Ligand: MAGNESIUM ION
  • Ligand: (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol -5-yl 4,6-O-[(1R)-ethylidene]-beta-D-glucopyranoside
KeywordsTopoisomerase / Etoposide / DNA / ISOMERASE / ISOMERASE-DNA complex
Function / homology
Function and homology information


positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / sister chromatid segregation / resolution of meiotic recombination intermediates / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / cellular response to ATP / positive regulation of double-strand break repair via nonhomologous end joining / DNA topological change / SUMOylation of DNA replication proteins / ribonucleoprotein complex binding ...positive regulation of single stranded viral RNA replication via double stranded DNA intermediate / sister chromatid segregation / resolution of meiotic recombination intermediates / DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) activity / DNA topoisomerase (ATP-hydrolysing) / cellular response to ATP / positive regulation of double-strand break repair via nonhomologous end joining / DNA topological change / SUMOylation of DNA replication proteins / ribonucleoprotein complex binding / forebrain development / axonogenesis / B cell differentiation / neuron migration / cellular response to hydrogen peroxide / cellular senescence / ribonucleoprotein complex / chromatin binding / nucleolus / DNA binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytosol
Similarity search - Function
DTHCT / DTHCT (NUC029) region / DNA topoisomerase 2, TOPRIM domain / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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 ...DTHCT / DTHCT (NUC029) region / DNA topoisomerase 2, TOPRIM domain / DNA topoisomerase II, eukaryotic-type / C-terminal associated domain of TOPRIM / C-terminal associated domain of TOPRIM / 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/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
DNA topoisomerase 2-beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsNaganuma M / Ehara H / Kim D / Nakagawa R / Cong A / Bu H / Jeong J / Jang J / Schellenberg MJ / Bunch H / Sekine S
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
CitationJournal: Nat Commun / Year: 2023
Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.
Authors: Heeyoun Bunch / Deukyeong Kim / Masahiro Naganuma / Reiko Nakagawa / Anh Cong / Jaehyeon Jeong / Haruhiko Ehara / Hongha Vu / Jeong Ho Chang / Matthew J Schellenberg / Shun-Ichi Sekine /
Abstract: The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. ...The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.
History
DepositionAug 5, 2022-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34022.png

Downloads & links

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Map

FileDownload / File: emd_34022.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.245 Å
Density
Contour LevelBy AUTHOR: 0.0125
Minimum - Maximum-0.02671669 - 0.06579608
Average (Standard dev.)0.00020672106 (±0.0026507408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_34022_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34022_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of human topoisomerase II beta with DNA and etoposide

EntireName: Ternary complex of human topoisomerase II beta with DNA and etoposide
Components
  • Complex: Ternary complex of human topoisomerase II beta with DNA and etoposide
    • Complex: human topoisomerase II beta
      • Protein or peptide: DNA topoisomerase 2-beta
    • Complex: DNA
      • DNA: 50-mer DNA
      • DNA: 50-mer DNA
  • Ligand: MAGNESIUM ION
  • Ligand: (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol -5-yl 4,6-O-[(1R)-ethylidene]-beta-D-glucopyranoside

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Supramolecule #1: Ternary complex of human topoisomerase II beta with DNA and etoposide

SupramoleculeName: Ternary complex of human topoisomerase II beta with DNA and etoposide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: human topoisomerase II beta

SupramoleculeName: human topoisomerase II beta / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: DNA topoisomerase 2-beta

MacromoleculeName: DNA topoisomerase 2-beta / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA topoisomerase (ATP-hydrolysing)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 183.54225 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAKSGGCGAG AGVGGGNGAL TWVTLFDQNN AAKKEESETA NKNDSSKKLS VERVYQKKTQ LEHILLRPDT YIGSVEPLTQ FMWVYDEDV GMNCREVTFV PGLYKIFDEI LVNAADNKQR DKNMTCIKVS IDPESNIISI WNNGKGIPVV EHKVEKVYVP A LIFGQLLT ...String:
MAKSGGCGAG AGVGGGNGAL TWVTLFDQNN AAKKEESETA NKNDSSKKLS VERVYQKKTQ LEHILLRPDT YIGSVEPLTQ FMWVYDEDV GMNCREVTFV PGLYKIFDEI LVNAADNKQR DKNMTCIKVS IDPESNIISI WNNGKGIPVV EHKVEKVYVP A LIFGQLLT SSNYDDDEKK VTGGRNGYGA KLCNIFSTKF TVETACKEYK HSFKQTWMNN MMKTSEAKIK HFDGEDYTCI TF QPDLSKF KMEKLDKDIV ALMTRRAYDL AGSCRGVKVM FNGKKLPVNG FRSYVDLYVK DKLDETGVAL KVIHELANER WDV CLTLSE KGFQQISFVN SIATTKGGRH VDYVVDQVVG KLIEVVKKKN KAGVSVKPFQ VKNHIWVFIN CLIENPTFDS QTKE NMTLQ PKSFGSKCQL SEKFFKAASN CGIVESILNW VKFKAQTQLN KKCSSVKYSK IKGIPKLDDA NDAGGKHSLE CTLIL TEGD SAKSLAVSGL GVIGRDRYGV FPLRGKILNV REASHKQIME NAEINNIIKI VGLQYKKSYD DAESLKTLRY GKIMIM TDQ DQDGSHIKGL LINFIHHNWP SLLKHGFLEE FITPIVKASK NKQELSFYSI PEFDEWKKHI ENQKAWKIKY YKGLGTS TA KEAKEYFADM ERHRILFRYA GPEDDAAITL AFSKKKIDDR KEWLTNFMED RRQRRLHGLP EQFLYGTATK HLTYNDFI N KELILFSNSD NERSIPSLVD GFKPGQRKVL FTCFKRNDKR EVKVAQLAGS VAEMSAYHHG EQALMMTIVN LAQNFVGSN NINLLQPIGQ FGTRLHGGKD AASPRYIFTM LSTLARLLFP AVDDNLLKFL YDDNQRVEPE WYIPIIPMVL INGAEGIGTG WACKLPNYD AREIVNNVRR MLDGLDPHPM LPNYKNFKGT IQELGQNQYA VSGEIFVVDR NTVEITELPV RTWTQVYKEQ V LEPMLNGT DKTPALISDY KEYHTDTTVK FVVKMTEEKL AQAEAAGLHK VFKLQTTLTC NSMVLFDHMG CLKKYETVQD IL KEFFDLR LSYYGLRKEW LVGMLGAEST KLNNQARFIL EKIQGKITIE NRSKKDLIQM LVQRGYESDP VKAWKEAQEK AAE EDETQN QHDDSSSDSG TPSGPDFNYI LNMSLWSLTK EKVEELIKQR DAKGREVNDL KRKSPSDLWK EDLAAFVEEL DKVE SQERE DVLAGMSGKA IKGKVGKPKV KKLQLEETMP SPYGRRIIPE ITAMKADASK KLLKKKKGDL DTAAVKVEFD EEFSG APVE GAGEEALTPS VPINKGPKPK REKKEPGTRV RKTPTSSGKP SAKKVKKRNP WSDDESKSES DLEETEPVVI PRDSLL RRA AAERPKYTFD FSEEEDDDAD DDDDDNNDLE ELKVKASPIT NDGEDEFVPS DGLDKDEYTF SPGKSKATPE KSLHDKK SQ DFGNLFSFPS YSQKSEDDSA KFDSNEEDSA SVFSPSFGLK QTDKVPSKTV AAKKGKPSSD TVPKPKRAPK QKKVVEAV N SDSDSEFGIP KKTTTPKGKG RGAKKRKASG SENEGDYNPG RKTSKTTSKK PKKTSFDQDS DVDIFPSDFP TEPPSLPRT GRARKEVKYF AESDEEEDDV DFAMFN

UniProtKB: DNA topoisomerase 2-beta

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Macromolecule #2: 50-mer DNA

MacromoleculeName: 50-mer DNA / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.449866 KDa
SequenceString:
(DG)(DA)(DG)(DT)(DC)(DG)(DC)(DG)(DA)(DG) (DA)(DG)(DA)(DT)(DC)(DC)(DC)(DA)(DG)(DC) (DG)(DC)(DG)(DC)(DA)(DG)(DA)(DA)(DC) (DT)(DT)(DG)(DG)(DG)(DG)(DA)(DG)(DC)(DC) (DG) (DC)(DC)(DG)(DC)(DC)(DG)(DC)(DC) (DA)(DT)

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Macromolecule #3: 50-mer DNA

MacromoleculeName: 50-mer DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.364773 KDa
SequenceString:
(DA)(DT)(DG)(DG)(DC)(DG)(DG)(DC)(DG)(DG) (DC)(DG)(DG)(DC)(DT)(DC)(DC)(DC)(DC)(DA) (DA)(DG)(DT)(DT)(DC)(DT)(DG)(DC)(DG) (DC)(DG)(DC)(DT)(DG)(DG)(DG)(DA)(DT)(DC) (DT) (DC)(DT)(DC)(DG)(DC)(DG)(DA)(DC) (DT)(DC)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,...

MacromoleculeName: (5S,5aR,8aR,9R)-9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol -5-yl 4,6-O-[(1R)-ethylidene]-beta-D-glucopyranoside
type: ligand / ID: 5 / Number of copies: 2 / Formula: EVP
Molecular weightTheoretical: 588.557 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 26067
FSC plot (resolution estimation)

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