Structural basis for regulation of a tripartite toxin-antitoxin system by dual phosphorylation

René Bærentsen, Stine Vang Nielsen, Jeppe Lyngsø, Francesco Bisiak, Jan Skov Pedersen, Kenn Gerdes, Michael Sørensen, Ditlev E. Brodersen

Research output: Working paper/Preprint Preprint


Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the E. coli hipBA system, HipA, is a kinase that inhibits inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli (EPEC) O127:H6 encodes an additional, tripartite TA module, hipBST, for which the HipT toxin was shown to specifically target tryptophanyl-tRNA synthetase, TrpS. Surprisingly, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is controlled remain poorly understood. Here, we show that HipBST is markedly different from HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, has evolved to function as antitoxin by breaking the kinase active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve to dually regulate binding of HipS and kinase activity. Finally, we demonstrate that the HipBST complex is dynamic and present a cohesive model for the regulation and activation of this type of three-component system.
Original languageEnglish
Number of pages76
Publication statusPublished - Jan 2022


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