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- EMDB-44766: Human DNA polymerase theta helicase domain in complex with inhibi... -

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Basic information

Entry
Database: EMDB / ID: EMD-44766
TitleHuman DNA polymerase theta helicase domain in complex with inhibitor AB25583, tetramer form
Map data
Sample
  • Complex: Human DNA polymerase theta helicase domain in complex with inhibitor AB25583
    • Protein or peptide: DNA polymerase theta
  • Ligand: (4P)-N-{5-[(4-chlorophenyl)methoxy]-1,3,4-thiadiazol-2-yl}-4-(2-methoxyphenyl)pyridine-3-carboxamide
KeywordsDNA repair / helicase / ATPase / TRANSFERASE-INHIBITOR complex
Function / homology
Function and homology information


single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis ...single-stranded DNA endodeoxyribonuclease activity / HDR through MMEJ (alt-NHEJ) / single-stranded DNA helicase activity / double-strand break repair via alternative nonhomologous end joining / replication fork processing / site of DNA damage / 5'-deoxyribose-5-phosphate lyase activity / somatic hypermutation of immunoglobulin genes / negative regulation of double-strand break repair via homologous recombination / error-prone translesion synthesis / DNA helicase activity / base-excision repair / protein homooligomerization / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / double-strand break repair / site of double-strand break / DNA helicase / damaged DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / DNA damage response / chromatin binding / Golgi apparatus / magnesium ion binding / ATP hydrolysis activity / nucleoplasm / ATP binding / identical protein binding / cytosol
Similarity search - Function
DNA polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain ...DNA polymerase theta-like, helix-turn-helix domain / : / Helix-turn-helix domain / DNA_pol_Q helicase like region helical domain / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA polymerase theta
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsIto F / Li Z / Chen XS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130889 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM.
Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Gurushankar Chandramouly / Mrityunjay Tyagi / Robert Betsch / John J Krais / Bernadette Taberi / Umeshkumar Vekariya / Marissa Calbert / Tomasz ...Authors: Fumiaki Ito / Ziyuan Li / Leonid Minakhin / Gurushankar Chandramouly / Mrityunjay Tyagi / Robert Betsch / John J Krais / Bernadette Taberi / Umeshkumar Vekariya / Marissa Calbert / Tomasz Skorski / Neil Johnson / Xiaojiang S Chen / Richard T Pomerantz /
Abstract: DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision ...DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
History
DepositionMay 7, 2024-
Header (metadata) releaseAug 28, 2024-
Map releaseAug 28, 2024-
UpdateAug 28, 2024-
Current statusAug 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44766.png

Downloads & links

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Map

FileDownload / File: emd_44766.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 400 pix.
= 368. Å		0.92 Å/pix.
x 400 pix.
= 368. Å		0.92 Å/pix.
x 400 pix.
= 368. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.28614202 - 0.58013964
Average (Standard dev.)0.00026360387 (±0.012879166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 368.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44766_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44766_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Human DNA polymerase theta helicase domain in complex with inhibi...

EntireName: Human DNA polymerase theta helicase domain in complex with inhibitor AB25583
Components
  • Complex: Human DNA polymerase theta helicase domain in complex with inhibitor AB25583
    • Protein or peptide: DNA polymerase theta
  • Ligand: (4P)-N-{5-[(4-chlorophenyl)methoxy]-1,3,4-thiadiazol-2-yl}-4-(2-methoxyphenyl)pyridine-3-carboxamide

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Supramolecule #1: Human DNA polymerase theta helicase domain in complex with inhibi...

SupramoleculeName: Human DNA polymerase theta helicase domain in complex with inhibitor AB25583
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 398 KDa

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Macromolecule #1: DNA polymerase theta

MacromoleculeName: DNA polymerase theta / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 99.802539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLLRRSGKR RRSESGSDSF SGSGGDSSAS PQFLSGSVLS PPPGLGRCLK AAAAGECKPT VPDYERDKLL LANWGLPKAV LEKYHSFGV KKMFEWQAEC LLLGQVLEGK NLVYSAPTSA GKTLVAELLI LKRVLEMRKK ALFILPFVSV AKEKKYYLQS L FQEVGIKV ...String:
MNLLRRSGKR RRSESGSDSF SGSGGDSSAS PQFLSGSVLS PPPGLGRCLK AAAAGECKPT VPDYERDKLL LANWGLPKAV LEKYHSFGV KKMFEWQAEC LLLGQVLEGK NLVYSAPTSA GKTLVAELLI LKRVLEMRKK ALFILPFVSV AKEKKYYLQS L FQEVGIKV DGYMGSTSPS RHFSSLDIAV CTIERANGLI NRLIEENKMD LLGMVVVDEL HMLGDSHRGY LLELLLTKIC YI TRKSASC QADLASSLSN AVQIVGMSAT LPNLELVASW LNAELYHTDF RPVPLLESVK VGNSIYDSSM KLVREFEPML QVK GDEDHV VSLCYETICD NHSVLLFCPS KKWCEKLADI IAREFYNLHH QAEGLVKPSE CPPVILEQKE LLEVMDQLRR LPSG LDSVL QKTVPWGVAF HHAGLTFEER DIIEGAFRQG LIRVLAATST LSSGVNLPAR RVIIRTPIFG GRPLDILTYK QMVGR AGRK GVDTVGESIL ICKNSEKSKG IALLQGSLKP VRSCLQRREG EEVTGSMIRA ILEIIVGGVA STSQDMHTYA ACTFLA ASM KEGKQGIQRN QESVQLGAIE ACVMWLLENE FIQSTEASDG TEGKVYHPTH LGSATLSSSL SPADTLDIFA DLQRAMK GF VLENDLHILY LVTPMFEDWT TIDWYRFFCL WEKLPTSMKR VAELVGVEEG FLARCVKGKV VARTERQHRQ MAIHKRFF T SLVLLDLISE VPLREINQKY GCNRGQIQSL QQSAAVYAGM ITVFSNRLGW HNMELLLSQF QKRLTFGIQR ELCDLVRVS LLNAQRARVL YASGFHTVAD LARANIVEVE VILKNAVPFK SARKAVDEEE EAVEERRNMR TIWVTGRKGL TEREAAALIV EEARMILQQ DLVEM

UniProtKB: DNA polymerase theta

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Macromolecule #2: (4P)-N-{5-[(4-chlorophenyl)methoxy]-1,3,4-thiadiazol-2-yl}-4-(2-m...

MacromoleculeName: (4P)-N-{5-[(4-chlorophenyl)methoxy]-1,3,4-thiadiazol-2-yl}-4-(2-methoxyphenyl)pyridine-3-carboxamide
type: ligand / ID: 2 / Number of copies: 4 / Formula: WCN
Molecular weightTheoretical: 452.913 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

Concentration0.8 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4500 / Average exposure time: 8.0 sec. / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000

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Image processing

Particle selectionNumber selected: 2932534
Startup modelType of model: INSILICO MODEL
In silico model: Generated by stochastic gradient descent using ab initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61097
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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