Crystal structure of Bacillus dafuensis Thoeris B1 protein in complex with NAD⁺

The Type 1 Thoeris defense system is an NAD⁺-based innate immune mechanism that protects bacterial populations against viral infection by triggering NAD⁺ depletion-induced cell death. Central to this system is the TIR domain-containing protein Ths B, which uses NAD⁺ to synthesize a cyclic ADPR (cADPR) signal upon sensing viral antigens. However, the structural basis of NAD⁺ binding by Ths B remains poorly understood. Here, we report the 1.54 Å resolution X-ray crystal structure of the Thoeris B1 protein from Bacillus dafuensis (Bd) in complex with NAD⁺. The structure reveals a canonical TIR fold comprising a five-stranded parallel β-sheet flanked by five α-helices, along with an unpredicted CCCH-type zinc finger domain formed by two flexible loops and a hydrophobic cavity. NAD⁺ binds in a distinctive C-shaped conformation, engaging residues near the conserved catalytic core. These findings suggest a pre-activation binding of NAD⁺ prior to antigen detection, providing structural clues into the specificity and catalytic mechanism of cADPR production. Our study uncovers unique structural features of bacterial TIR domains and expands our understanding of the molecular basis of Thoeris-mediated antiviral immunity.