Mechanistic insights into target searching, de-dimerization, and activation of Mucilaginibacter paludis Argonaute

Argonaute proteins are found in both eukaryotes and prokaryotes, participating in defense and gene regulation. Although extensive structural studies have elucidated guide loading, target recognition and catalytic mechanisms of long prokaryotic Argonautes (pAgos), the structural basis underlying target searching remains largely unexplored. Here, we report cryo-electron microscopy structures of the RNA-preferring MbpAgo from Mucilaginibacter paludis in guide-bound (3.2 Å) and guide–target-bound (3.1 Å) states. Unexpectedly, guide loading promotes the formation of a symmetric dimer that sandwiches an additional nucleic acid fragment at the dimer interface, revealing a surveillance configuration for candidate target sampling. Progressive guide-target duplex propagation beyond ~12 bp induces pronounced conformational rearrangements, including PAZ domain rotation and inversion of an interlocked PIWI loop, thereby disrupting the dimer interface and generating of a catalytically competent monomer. Stabilization of the dimer through engineered disulfide bonds suppresses cleavage activity, demonstrating that target-triggered de-dimerization is required for activation. Together, our findings uncover a structural framework for target searching in an RNA-preferring pAgo and uncover a monomer-dimer-monomer activation trajectory in which guide-induced dimerization is followed by target-triggered de-dimerization, establishing assembly-state remodeling as a regulatory principle in pAgo-mediated nucleic acid recognition.