CHARACTERIZATION OF THE COVALENT BINDING OF THIOSTREPTON TO A THIOSTREPTON-INDUCED PROTEIN FROM STREPTOMYCES-LIVIDANS

Thiostrepton is a highly modified multicyclic peptide antibiotic synth esized by diverse bacteria. Although best known as an inhibitor of pro tein synthesis, thiostrepton is also a potent activator of gene expres sion in Streptomyces lividans. In these studies, we characterize the n ature of the interaction between thiostrepton and two proteins that it induces, TipAL and TipAS. In the absence of added cofactors, thiostre pton 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-thio strepton complex as determined by both sodium dodecyl sulfate-polyacry lamide,eel electrophoresis (SDS-PAGE) and mass spectrometry (MS) was e quivalent to the sum of TipAS and thiostrepton. Thiostrepton also reac ted spontaneously with free cysteine (but not with other amino acids t ested) to generate stable compounds having masses equivalent to thiost repton plus 3 or 4 cysteines. Blocking experiments indicated that comp lex formation required dehydroalanine residues on thiostrepton and cys teine residues on TipAS. When the TipAS-thiostrepton complex was diges ted with trypsin and analyzed by MS, the thiostrepton adduct was found bound only to the unique cysteine-containing TipAS peptide fragment. Amino acid analysis confirmed that the TipAS-thiostrepton complex cont ained lanthionine, the product of a reaction between dehydroalanine an d cysteine. Together, these data document a covalent attachment of thi ostrepton to TipA proteins mediated by bond formation between dehydroa lanine of thiostrepton and cysteine of TipAS. Implications regarding t he function of TipAS as a thiostrepton (electrophile)-sequestering pro tein and thiostrepton-mediated activation of TipAL as a model of irrev ersible transcriptional activation are discussed.