Publications by authors named "Arnaud Vanden Broeck"

7 Publications

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Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex.

Nat Commun 2019 10 30;10(1):4935. Epub 2019 Oct 30.

Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67404, Illkirch Cedex, France.

DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.
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http://dx.doi.org/10.1038/s41467-019-12914-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821735PMC
October 2019

Structural Basis for DNA Gyrase Interaction with Coumermycin A1.

J Med Chem 2019 04 3;62(8):4225-4231. Epub 2019 Apr 3.

Integrated Structural Biology Department, IGBMC, UMR7104 CNRS, U1258 Inserm, University of Strasbourg, Illkirch 67404 , France.

Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01928DOI Listing
April 2019

Biochemical and structural characterization of a mannose binding jacalin-related lectin with two-sugar binding sites from pineapple (Ananas comosus) stem.

Sci Rep 2018 07 31;8(1):11508. Epub 2018 Jul 31.

Laboratory of crystallography, Center for Protein Engineering-InBioS, B5a, University of Liège, 4000, Liège, Belgium.

A mannose binding jacalin-related lectin from Ananas comosus stem (AcmJRL) was purified and biochemically characterized. This lectin is homogeneous according to native, SDS-PAGE and N-terminal sequencing and the theoretical molecular mass was confirmed by ESI-Q-TOF-MS. AcmJRL was found homodimeric in solution by size-exclusion chromatography. Rat erythrocytes are agglutinated by AcmJRL while no agglutination activity is detected against rabbit and sheep erythrocytes. Hemagglutination activity was found more strongly inhibited by mannooligomannosides than by D-mannose. The carbohydrate-binding specificity of AcmJRL was determined in some detail by isothermal titration calorimetry. All sugars tested were found to bind with low affinity to AcmJRL, with K values in the mM range. In agreement with hemagglutination assays, the affinity increased from D-mannose to di-, tri- and penta-mannooligosaccharides. Moreover, the X-ray crystal structure of AcmJRL was obtained in an apo form as well as in complex with D-mannose and methyl-α-D-mannopyranoside, revealing two carbohydrate-binding sites per monomer similar to the banana lectin BanLec. The absence of a wall separating the two binding sites, the conformation of β7β8 loop and the hemagglutinating activity are reminiscent of the BanLec His84Thr mutant, which presents a strong anti-HIV activity in absence of mitogenic activity.
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http://dx.doi.org/10.1038/s41598-018-29439-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068142PMC
July 2018

Publisher Correction: Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Sci Rep 2018 Jul 10;8(1):10673. Epub 2018 Jul 10.

Université de Strasbourg, CNRS UMR7104, INSERM U1258, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-28936-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039497PMC
July 2018

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Sci Rep 2018 06 18;8(1):9272. Epub 2018 Jun 18.

Université de Strasbourg, CNRS UMR7104, INSERM U1258, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.
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http://dx.doi.org/10.1038/s41598-018-27606-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006247PMC
June 2018

A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP.

PLoS One 2015 13;10(10):e0140082. Epub 2015 Oct 13.

Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Sart Tilman, Belgium.

In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140082PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604126PMC
June 2016