Dual-inhibitory mechanism of the bacterial retron Ec78 antiphage defense system

Retrons are prokaryotic defense modules that protect bacteria from phage infection through abortive infection. The retron Ec78 system employs a two-component effector complex PtuAB to execute this defense. Despite recent advances in structural research, the molecular mechanism by which PtuAB effector is regulated remains unknown. Here, we reveal that PtuAB is subject to a dual-inhibitory mechanism mediated by ATP/ADP and the RT-msDNA antitoxin. ATP/ADP binds nucleotide-binding domain (NBD) of PtuAB and induces the assembly of an inactive tetrameric complex, whereas the RT-msDNA stabilizes an inhibited conformation of Ec78 complex and stimulates ATP turnover to prime PtuAB for rapid activation. Structural analyses show that RT-msDNA dissociation and nucleotide release from PtuA induce conformational rearrangements in the NBD of PtuA and a downward displacement of a key β-loop-β motif, driving disassembly of the PtuAB tetramer through an allosteric mechanism and thereby activating its tRNA^Tyr cleavage activity. Our findings uncover how nucleotides-specifically ATP and ADP-regulate the activity of this abortive infection system, and establish a dual-inhibition model of retron Ec78 system, expanding the understanding of the regulation mechanism of PtuAB activation in prokaryotic immune systems.