Mishra, B. P.; Masic, V.; Mosaiab, T.; Belani, H.; Conroy, T.; Brillault, L.; Swarbrick, C. M. D.; Zang, Z.; Kobe, B.; Shi, Y.; von Itzstein, M.; Gerdt, J. P.; Ve, T.

Thoeris defence systems protect bacteria from phages via abortive infection. In type I Thoeris systems, ThsA effectors containing silent information regulator 2 (SIR2) and SMF/DprA-LOG (SLOG) domains are activated by the cyclic ADP-ribose (ADPR) isomer 3'cADPR, triggering abortive infection via nicotinamide adenine dinucleotide (NAD+) depletion. 3'cADPR activates the NADase activity of Bacillus subtilis ThsA tetramers via filament formation of its SIR2 domains, but the molecular details of how 3'cADPR triggers this process remain incompletely understood. Here, we demonstrate that ThsA activation by 3'cADPR-induced SIR2 filament formation is conserved in type I Thoeris systems from Streptococcus equi and Entercoccus facium. We present cryo-electron microscopy structures of the S. equi ThsA filament bound to 3'cADPR and the non-cleavable NAD+ analog carba-NAD+, and of the S. equi ThsA tetramer bound to 3'cADPR. These structures reveal that SIR2 filament formation is required to stabilise an active site conformation that can bind and hydrolyse NAD+. The structures also show that 3'cADPR induces quaternary alterations in the SLOG dimers and consequently the SIR2 tetramer to enable ThsA filament formation. Collectively, our study provides a comprehensive understanding of 3'cADPR-induced activation of type I Thoeris effectors.