Distinct mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity

Prokaryotic Argonautes (pAgos) are widely distributed and provide immunity against invading DNA. Based on their domain architecture, pAgos are classified into three major groups: long-A, long-B, and short pAgos. Among them, long-B pAgos remain the least understood subgroup. Here, we show that a long-B pAgo-nuclease system ( Ec BPAN) from Escherichia coli provides the first clear evidence of anti-phage activity in long-B pAgo systems. By combining structural determination, biochemical analyses, and in vivo phage-resistance assays, we elucidated the activation mechanism of Ec BPAN. We found that RNA-guided Ec Ago recognizes target DNA and subsequently recruits the autoinhibited dimer of its associated nuclease ( Ec bAgaN) to form an unprecedented 8:8 pAgo-nuclease complex with robust nonspecific DNase activity. The cryo-EM structure of the activated complex revealed a distinctive bowl-shaped architecture in which the C-terminal nuclease domains of Ec bAgaN form an active octamer, while the N-terminal domains engage four Ec Ago-guide RNA-target DNA ternary dimers in an interleaved manner, thereby relieving autoinhibition. Together, these findings provide the first mechanistic insights into a long-B pAgo defense system and reveal a mode of immune activation fundamentally distinct from those of long-A and short pAgo systems.