Characterization of the Covalent Binding of Thiostrepton to a Thiostrepton-Induced Protein from Streptomyces lividans†

Thiostrepton is a highly modified multicyclic peptide antibiotic synthesized by diverse bacteria. Although best known as an inhibitor of protein synthesis, thiostrepton is also a potent activator of gene expression in Streptomyces lividans. In these studies, we characterize the nature of the interaction between thiostrepton and two proteins that it induces, TipAL and TipAS. In the absence of added cofactors, thiostrepton formed a complex with either TipAL or TipAS in aqueous solution. The TipA−thiostrepton complex was not dissociated by denaturants such as SDS, urea, or disulfide reducing agents. The mass of the TipAS−thiostrepton complex as determined by both sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and mass spectrometry (MS) was equivalent to the sum of TipAS and thiostrepton. Thiostrepton also reacted spontaneously with free cysteine (but not with other amino acids tested) to generate stable compounds having masses equivalent to thiostrepton plus 3 or 4 cysteines. Blocking e...