Structural and functional characterization of SPARTA system

Short prokaryotic Argonaute associated with TIR-APAZ (SPARTA) is a bacterial defense system that oligomerizes to activate the TIR domain, depleting NAD⁺ upon guide-mediated target DNA recognition. Previous studies have shown a marked activity divergence between thermophilic Crenotalea thermophila SPARTA (CrtSPARTA) and Maribacter polysiphoniae SPARTA (MapSPARTA); however, the underlying mechanism remains unclear. Here, through biochemical and structural analysis, we found that compared with the relatively rigid CrtSPARTA, MapSPARTA exhibits much more flexibility in its TIR domain, which flips during the formation of active tetramers. Interestingly, we found that the activity of CrtSPARTA, but not MapSPARTA, could be significantly enhanced when we weakened the interaction between the MID and TIR domains by introducing mutations at the interface, suggesting that the MID-TIR interaction restricts the release of CrtTIR and thus the activation of CrtSPARTA. Following guide-target recognition, this flexibility-induced activation can be further promoted by higher temperatures within the physiological range or Ca²⁺, together suggesting that the restriction of TIR by the MID-TIR interaction may be a strategy to prevent auto-activation of thermophilic CrtSPARTA. Meanwhile, we also found that tRNA fragments can serve as guide RNAs for SPARTA activation, which probably reveals the origin of the RNA guide for prokaryotic Argonaute. This also suggests the possibility that SPARTA may function cooperatively with other defense systems involving a tRNA endonuclease to efficiently respond to viral infection.