Insights into the Neutralization and DNA Binding of Toxin-Antitoxin System ParE_SO-CopA_SO by Structure-Function...

ParE_SO-CopA_SO is a new type II toxin-antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParE_SO severely inhibits cell growth, while CopA_SO functions as an antitoxin to neutralize ParE_SO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParE_SO-CopA_SO complex that adopted an open V-shaped heterotetramer with the organization of ParE_SO-(CopA_SO)₂-ParE_SO. The structure showed that upon ParE_SO binding, the intrinsically disordered C-terminal domain of CopA_SO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParE_SO. Thermodynamics analysis showed the DNA-binding affinity of CopA_SO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParE_SO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParE_SO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParE_SO-CopA_SO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.