Fig. 6: The competitive binding of DSAD1 and TTP to DSR2 effectively inhibits its NADase activity.

a NAD⁺ hydrolase activities of DSR2 or DSR2-DSAD1 complex in the presence of different amount of TTP. All assays were performed at least in triplicate (mean ± SD, n = 3 independent replicates), and the standard deviations were calculated using GraphPad Prism. b NAD⁺ hydrolase activities of DSR2-TTP complex in the presence of different amount of DSAD1 protein. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). c Strep pull down assay was performed to analyze the direct competition between DSAD1 and TTP for binding to DSR2. In this assay, DSR2 was pre-incubated with different amount of DSAD1 or TTP for 1 h, and then, the third protein was added to the reaction mixtures for evaluate the competitive binding to DSR2. The experiment was replicated independently three times, producing similar results. d Superimposed ribbon diagrams of the DAR2 apo, DSR2-TTP and DSR2-DSAD1 complex. DSR2 dimers in the DSR2-DSAD1 complex, DSR2-TTP complex and apo DSR2 are colored in cyan, magenta and gray, respectively. TTP and DSAD1 are colored in wheat and orange, respectively. The key residues required for NAD⁺ coordination but shifted relative to the other protomer are shown as sticks. e A proposed model to illustrate the mechanisms of DSR2 activation upon phages infection and phages immune evasion against the DSR2-dependent defense system.