Interactions between the toxin Kid of the Bacterial parD system and the antitoxins Kis and MazE.

摘要

The proteins Kid and Kis are thetoxin and antitoxin, respectively, encoded by theparD operon of Escherichia coli plasmid R1. Kis preventsthe inhibition of E. coli cell growth caused bythe RNA cleavage activity of Kid. Overproduction ofMazE, the chromosome-encoded homologue of Kis,has been demonstrated to neutralize Kid toxicity toa certain extent in the absence of native Kis. Here,we show that a high structural similarity exists betweenthese antitoxins, using NMR spectroscopy. Wereport about the interactions between Kid and Kisthat are responsible for neutralization of Kid toxicityand enhance autoregulation of parD transcription.Native macromolecular mass spectrometrydata demonstrate that Kid and Kis form multiplecomplexes. At Kis:Kid ratios equal to or exceeding1:1, as found in vivo in a plasmid-containing cell,various complexes are present, ranging from Kid2–Kis2 tetramer up to Kis2–Kid2–Kis2–Kid2–Kis2 decamer.When Kid is in excess of Kis, corresponding toan in vivo situation immediately after loss of theplasmid, the Kid2–Kis2–Kid2 heterohexamer is themost abundant species. NMR chemical shift and intensityperturbations in the 1H 15N HSQC spectra ofKid and Kis, observed when titrating the partnerprotein, show that the interaction sites of Kid andKis resemble those within the previously reportedMazF2–MazE2–MazF2 complex. Furthermore, we demonstratethat Kid2–MazE2 tetramers can be formedvia weak interactions involving a limited part of theKis-binding residues of Kid. The functional roles ofthe identified Kid–Kis and Kid–MazE interactionsites and complexes in toxin neutralization andrepression of transcription are discussed.