Toxin dimerization and a distinct DNA-binding architecture define chromosomal Phd-Doc regulation.

Toxin-antitoxin (TA) systems are widespread bacterial regulatory modules, yet the structural diversity of chromosomally encoded TA regulation remains incompletely understood. Here, we show that the Enterococcus faecalis Phd-Doc module adopts a regulatory architecture distinct from the canonical Escherichia coli paradigm. Structural analyses reveal that toxin neutralization is associated with antitoxin-mediated toxin dimerization rather than direct occlusion of the conserved catalytic motif. In parallel, the N-terminal domain of E. faecalis Phd forms a β-sheet-based DNA-binding arrangement that recognizes a single palindromic operator, contrasting with the cooperative dual-operator recognition observed in E. coli. Together, these features define an alternative configuration for operator recognition and toxin regulation within a chromosomal TA system. Structure-guided peptide design further demonstrates that the E. faecalis Phd-Doc interface can be selectively perturbed, highlighting the potential of exploiting distinct TA architectures for differential functional modulation.