The structural basis of DSAD1-DSR2 mediated phage immune evasion

Abstract

Abstract DSR2 belongs to the family of defense-associated sirtuins (DSRs) which protect bacteria from phage infection by hydrolyzing NAD+. Phage propagation is effectively inhibited through the induction of abortive infection by depleting this essential molecule. The enzymatic activity of DSR2 is triggered by the tail tube protein of phages, enabling its functional activation. To escape bacterial immunity, certain phages have evolved to encode DSAD1 protein, which binds to and deactivates DSR2, neutralizing its defensive capabilities. However, the structural basis underlying the degradation of NAD+ by DSR2 and the inhibitory mechanism of its NADase activity by DSAD1 remain elusive. Here, we report the cryo-EM structures of apo DSR2 from Bacillus subtilis and the DSR2-DSAD1 complex. The apo structure reveals that DSR2 assembles into a head-to-head tetramer, which is mediated by its N-terminal Sir2 domain. DSR2 Sir2 domain possesses the highly conserved residues that have been found to be essential for NAD+ coordination and depletion in ThsA. The structures of DSR2-DSAD1 complex demonstrate the binding of two DSAD1 molecules to the two of four C-terminal helical regions of DSR2 tetramer, forming a hetero-hexameric complex. Two DSAD1 protomers locate separately at two ends of DSR2 tetramer either on the same side or opposite sides. This binding at DSR2 C-terminus induces a conformational change in the Sir2 domain at its N-terminus, potentially suppressing its enzymatic activity. These findings not only broaden our understanding of the DSR2-mediated anti-phage defense system, but also uncover the mechanism of DSAD1-dependent phage immune evasion.