Toxin–antitoxin genes play important roles in the regulation of bacterial growth during stress. One response to stress is selective proteolysis of antitoxin proteins which releases their cognate toxin partners causing rapid inhibition of growth. The features of toxin–antitoxin complexes that are important to inhibit toxin activity as well as to release the active toxin remain elusive. Furthermore, it is unclear how antitoxins are selected for proteolysis by cellular proteases. Here, we test the minimal structural requirements of the Escherichia coli DinJ antitoxin to suppress its toxin partner, YafQ. We find that DinJ–YafQ complex formation is critically dependent on the last ten C-terminal residues of DinJ. However, deletion of these 10 DinJ residues has little effect on transcriptional autorepression suggesting that the YafQ toxin is not a critical component of the repression complex in contrast to other toxin–antitoxin systems. We further demonstrate that loop 5 preceding these ten C-terminal residues is important for Lon-mediated proteolysis. These results provide important insights into the critical interactions between toxin–antitoxin pairs necessary to inhibit toxin activity and the regulated proteolysis of antitoxins.
Bacterial toxin–antitoxin complexes control growth dependent upon the cellular state with stress-induced antitoxin proteolysis leading to release of the cognate toxin to inhibit growth. We demonstrate that the C-terminus of Escherichia coli DinJ antitoxin is critical for interactions with the YafQ toxin and additionally identify the DinJ protein C-terminus as the recognition site of Lon protease. These findings reveal a mechanism of coordinated proteolysis and release of the YafQ toxin.