title: Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE.
abstract: The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.
authors.affiliation: Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Utrecht, The Netherlands.
authors.forename: Monique B
authors.initials: MB
authors.lastname: Kamphuis
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Monti
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Sandra
authors.initials: S
authors.lastname: Santos-Sierra
authors.forename: Gert E
authors.initials: GE
authors.lastname: Folkers
authors.forename: Marc
authors.initials: M
authors.lastname: Lemonnier
authors.forename: Ramón
authors.initials: R
authors.lastname: Díaz-Orejas
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Rolf
authors.initials: R
authors.lastname: Boelens
mesh_terms: D000992:Antitoxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009876:Operon; D010957:Plasmids / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C068353:Kid protein, Bacteria; C068354:Kis protein, Bacteria; C422954:MazE protein, E coli; C489746:ParD protein, E coli