Fig. 1: DSR2 assembles into a rod-like head-to-head tetramer.

a Domain organization of DSR2 from Bacillus subtilis 29 R, activator TTP from SPR phage and inhibitor protein DSAD1 from SPbeta phage. b NAD⁺ hydrolase activities of DSR2 alone or in the presence of TTP and DSAD1. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). c Cryo-EM density map of DSR2 tetramer in side view. The Sir2 domain tetramer is colored in cyan, while the helical repeats domain in the DSR2 dimer was colored in forest green and magenta. d Ribbon diagram of the DSR2 tetramer, and the domain was colored as in (a) and (c). e Electron density of the Sir2 domain of DSR2 docked with the Sir2 model. f Ribbon diagram of the Sir2 domain of DSR2. g, h Overlay of structures of the Sir2 domains of DSR2 and ADPR-bound ThsA N112A (PDB ID: 8BTP) or NAD⁺-bound Sir2-HerA system (PDB ID: 8UAF). DSR2, ThsA and Sir2 are colored in cyan, wheat and light pink, respectively. i Close-up view of the NAD⁺-binding pockets of the Sir2 domains from DSR2, ThsA and Sir2-HerA system. The key residues for NAD⁺ coordination are shown as sticks. j NAD⁺ hydrolase activities of WT DSR2 or its mutants in the presence of TTP. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). k Ribbon diagram of the DSR2 dimer in one side of the tetramer. The DSR2 helical region is divided into a helix-turn-helix (HTH) domain, a helix bundle domain, a long HEAT repeat domain and a C-terminal domain. Two partner-binding cavities (one opened cavity and one closed cavity) are constituted by the HEAT repeats and C-terminal domains of DSR2 dimer. l The opened cavity protomer of DSR2 aligned to the closed cavity promoter. Compared with the closed cavity, the helix of C-terminal domain with the opened cavity exhibits an outward twist.