Structural basis for kinase inhibition in the tripartiteE. coliHipBST toxin-antitoxin system

Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of theE. coli hipBAsystem, HipA, is a kinase that inhibits translation via phosphorylation of glutamyl-tRNA synthetase. EnteropathogenicE. coli(EPEC) O127:H6 encodes thehipBA-like, tripartite TA system;hipBST, in which the HipT toxin specifically targets the tryptophanyl-tRNA synthetase, TrpS. Notably, in the tripartite system, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is inhibited remain poorly understood. Here, we show that HipBST is structurally different fromE. coliHipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, inhibits the kinase through insertion of a conserved Trp residue into the active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve different roles and affect the ability of HipS to neutralize HipT. Finally, solution structural studies show how phosphorylation affects overall TA complex flexibility.