Interactions between the toxin kid of the bacterial parD system and the antitoxins Kis and MazE

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 Kid2-Kis2 tetramer up to Kis2-Kid2-Kis2-Kid2-Kis2 decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid2-Kis2-Kid2 heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the 1H 15N 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 MazF2-MazE2-MazF2 complex. Furthermore, we demonstrate that Kid2-MazE2 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.