Nucleic acid-triggered NADase activation of a short prokaryotic Argonaute.

Argonaute (Ago) mediates RNA or DNA guided inhibition of nucleic acids 1,2 . Although the mechanisms underlying eukaryotic (eAgos) and long prokaryotic Ago (pAgos) proteins are known, that of short pAgos remains elusive. Here, we determined cryo-EM structures of short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA (3.27 Å), a guide RNA / target DNA loaded Crt-SPARTA (3.27 Å), two Crt-SPARTA dimers with distinct TIR organization (3.49 Å and 3.50 Å), and a Crt-SPARTA tetramer (3.41 Å). These structures reveal that the Crt-SPARTA is composed of a bilobal-fold Ago lobe connecting with a TIR lobe. Whereas the Crt-Ago harbors a MID and a PIWI domains, Crt-TIR-APAZ harbors a TIR, a N-like, a Linker and a Trigger domains. The bound RNA/DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the Trigger acts as a roadblock preventing the guide RNA 5'- and the target DNA 3'-ends from reaching their canonical pockets, which disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA/DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR assemble into two parallel, head-to-tail TIR organization, indicating a NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short pAgos in defensing invading nucleic acids and provide insights for optimizing SPARTA-based programmable DNA sequences detection.