title: Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.
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.
authors.affiliation: Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Freie Universit&#228;t Berlin, K&#246;nigin-Luise-Str. 2+4, 14195 Berlin, Germany. isabelfernandezb77@yahoo.es.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Fernández-Bachiller
authors.affiliation: Institute of Biochemistry and Biophysics, PAS, Pawi&#324;skiego 5a, 02-106 Warszawa, Poland. iwonab@ibb.waw.pl.
authors.forename: Iwona
authors.initials: I
authors.lastname: Brzozowska
authors.affiliation: Institute of Biochemistry and Biophysics, PAS, Pawi&#324;skiego 5a, 02-106 Warszawa, Poland. nodolczyk@ibb.waw.pl.
authors.forename: Norbert
authors.initials: N
authors.lastname: Odolczyk
authors.affiliation: Institute of Biochemistry and Biophysics, PAS, Pawi&#324;skiego 5a, 02-106 Warszawa, Poland. ulazet@ibb.waw.pl.
authors.forename: Urszula
authors.initials: U
authors.lastname: Zielenkiewicz
authors.affiliation: Institute of Biochemistry and Biophysics, PAS, Pawi&#324;skiego 5a, 02-106 Warszawa, Poland. piotr@ibb.waw.pl.|Faculty of Biology, Warsaw University, Miecznikowa 1, 02-106 Warszawa, Poland. piotr@ibb.waw.pl.
authors.forename: Piotr
authors.initials: P
authors.lastname: Zielenkiewicz
authors.affiliation: Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Freie Universit&#228;t Berlin, K&#246;nigin-Luise-Str. 2+4, 14195 Berlin, Germany. joerg.rademann@fu-berlin.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Rademann
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000992:Antitoxins / Q000378:metabolism*; D001419:Bacteria / Q000187:drug effects / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D015195:Drug Design; D024901:Drug Resistance, Multiple, Bacterial; D005454:Fluorescence Polarization; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D000992:Antitoxins; D001427:Bacterial Toxins; D008024:Ligands; D010455:Peptides
keywords: bacterial resistance; drug discovery; fluorescence polarization; protein–protein interactions; toxin–antitoxin system