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Toxins 2016, 8(7), 222; doi:10.3390/toxins8070222

Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

1
Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany
2
Institute of Biochemistry and Biophysics, PAS, Pawińskiego 5a, 02-106 Warszawa, Poland
3
Faculty of Biology, Warsaw University, Miecznikowa 1, 02-106 Warszawa, Poland
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Anton Meinhart
Received: 14 May 2016 / Revised: 29 June 2016 / Accepted: 5 July 2016 / Published: 16 July 2016
(This article belongs to the Special Issue Toxin-Antitoxin System in Bacteria)
View Full-Text   |   Download PDF [4352 KB, uploaded 16 July 2016]   |  

Abstract

Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. View Full-Text
Keywords: protein–protein interactions; toxin–antitoxin system; drug discovery; bacterial resistance; fluorescence polarization protein–protein interactions; toxin–antitoxin system; drug discovery; bacterial resistance; fluorescence polarization
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Fernández-Bachiller, M.I.; Brzozowska, I.; Odolczyk, N.; Zielenkiewicz, U.; Zielenkiewicz, P.; Rademann, J. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization. Toxins 2016, 8, 222.

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