Self-association activates ThsB NAD hydrolase for defense against phage infection.

Toll/interleukin-1 receptor (TIR) domain serves as a canonical component in both animal and plant innate immunity pathways and is indicated, in some cases, to mediate nicotinamide adenine dinucleotide (NAD) cleavage via self-association. Recent studies have revealed the involvement of TIR domains in a bacterial anti-phage defense system called Thoeris. The Thoeris system consists of two core proteins, ThsA and ThsB. Phage infection triggers the TIR-containing ThsB to produce an isomer of cyclic ADP-ribose, which is then transferred to and activates ThsA, leading to NAD depletion and subsequent cell death. However, the mechanism of ThsB activation remains elusive. Here, we present high-resolution crystal structures of E. coli ThsA and ThsB. Notably, an intact NAD molecule is observed in the active site of ThsB, implying that monomeric ThsB does not possess NADase activity. We demonstrate that ThsB forms 7-fold oligomers through negative staining electron microscopy, suggesting that self-association activates ThsB NAD hydrolase. Our findings indicate a new TIR self-association assembly in bacterial anti-phage systems.