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. 2024 Mar 27;15(1):2692.
doi: 10.1038/s41467-024-47030-z.

Insights into the modulation of bacterial NADase activity by phage proteins

Hang Yin #  1   2 , Xuzichao Li #  1   3 , Xiaoshen Wang #  1   3 , Chendi Zhang #  4 , Jiaqi Gao  1   3 , Guimei Yu  1 , Qiuqiu He  1   3 , Jie Yang  1 , Xiang Liu  5 , Yong Wei  6 , Zhuang Li  7 , Heng Zhang  8   9
Affiliations

Insights into the modulation of bacterial NADase activity by phage proteins

Hang Yin et al. Nat Commun. .

Abstract

The Silent Information Regulator 2 (SIR2) protein is widely implicated in antiviral response by depleting the cellular metabolite NAD+. The defense-associated sirtuin 2 (DSR2) effector, a SIR2 domain-containing protein, protects bacteria from phage infection by depleting NAD+, while an anti-DSR2 protein (DSR anti-defense 1, DSAD1) is employed by some phages to evade this host defense. The NADase activity of DSR2 is unleashed by recognizing the phage tail tube protein (TTP). However, the activation and inhibition mechanisms of DSR2 are unclear. Here, we determine the cryo-EM structures of DSR2 in multiple states. DSR2 is arranged as a dimer of dimers, which is facilitated by the tetramerization of SIR2 domains. Moreover, the DSR2 assembly is essential for activating the NADase function. The activator TTP binding would trigger the opening of the catalytic pocket and the decoupling of the N-terminal SIR2 domain from the C-terminal domain (CTD) of DSR2. Importantly, we further show that the activation mechanism is conserved among other SIR2-dependent anti-phage systems. Interestingly, the inhibitor DSAD1 mimics TTP to trap DSR2, thus occupying the TTP-binding pocket and inhibiting the NADase function. Together, our results provide molecular insights into the regulatory mechanism of SIR2-dependent NAD+ depletion in antiviral immunity.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Cryo-EM structure of DSR2 in complex with DSAD1.
a Domain organization of the DSR2 and DSAD1 proteins. b Cryo-EM structure of the DSR2-DSAD1 binary complex. The N-terminal SIR2 domain, Lid subdomain, and C-terminal H1, H2, H3, and H4 subdomains are colored in wheat, purple, magenta, cyan, green and blue, respectively. The DSAD1 protein is colored in orange. The lid region of the SIR2 domain responsible for the inter-dimer interaction is marked in the red box. The schematic diagram in the gray box shows the binding mode of DSAD1 to DSR2 molecules. c Overall structure of a single DSR2 molecule indicates a fishhook-like architecture (gray box). The catalytic pocket within DSR2 is marked in a black circle. The same color scheme as in (a) is used. d Detailed insights into the inter-dimer interface formed by the SIR2 domains. Key residues involved in the inter-dimer interaction are shown in stick representation.
Fig. 2
Fig. 2. The dimerization of CTD is required for DSAD1 binding.
a Close-up view of DSAD1 binding site on the C-terminal domain (CTD) of DSR2. The DSR2-A molecule is colored in the same scheme as in Fig. 1a, and the DSR2-B molecule is colored in gray for clarity. The DSAD1 protein is colored in orange. b Detailed insights into the interaction between DSAD1 and the H3 subdomain of DSR2. Key interacting residues are shown in stick representation. c Detailed insights into the interaction between DSAD1 protein and the H3 and H4 subdomains of DSR2 dimer. Key interacting residues are shown in stick representation. d In vitro pull-down of wild-type (WT) DSR2 and mutants by His-tagged DSAD1. ΔH4 indicates the deletion of H4 subdomain (aa 1–860). The K960A/D993A mutation had little impact on the DSR2-DSAD1 association. The gel represents three independent replicate experiments. Source data are provided as a Source data file.
Fig. 3
Fig. 3. Cryo-EM structures of TTP alone and in complex with DSR2.
a In vitro NAD+ degradation assays of the full-length DSR2 protein and SIR2 domain-only protein in the presence or absence of TTP. The N133A/H171A catalytic mutant of the DSR2 protein was used as the negative control. 50 μM ɛ-NAD+ was used as the substrate. Only the WT DSR2 protein can degrade NAD+ in the presence of the TTP protein. The experiment was replicated three times. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). b In vitro pull-down of wild-type (WT) DSR2 and mutants by His-tagged TTP. The gel represents three independent replicate experiments. ΔSIR2 indicates the deletion of the SIR2 subdomain (aa 304–1005), and SIR2 indicates a SIR2 domain-only protein (aa 1–303). The experiment was replicated three times. c Atomic model of the TTP tube (upper panel), each layer of the TTP ring is colored in wheat, gray, teal, and light blue, respectively. The lower panel shows the structure of TTP subunit in the TTP tube. The single copy of the TTP subunit is shown as cartoon representations and colored in yellow. BS1, β-sandwich domain 1; BS2, β-sandwich domain 2. d Cryo-EM structure of DSR2-TTP complex in state 1. The same color scheme as in Fig. 1a is adopted. The four TTP molecules are colored in yellow. e Cryo-EM structure of DSR2-TTP complex in state 2. The SIR2 domains are missing in this state. f Structural superposition of TTP proteins in the DSR2-TTP complex (yellow) and TTP tube (gray). A shift in the α1 helix of TTP is indicated by a blue arrow. Source data are provided as a Source data file.
Fig. 4
Fig. 4. Interactions between DSR2 and TTP proteins.
a Close-up view of the interactions between the BS1 domain of TTP protein and DSR2 proteins. The same color scheme as in Fig. 1a is used for the DSR2-A molecule. The DSR2-B molecule is colored in gray and the bound TTP is colored in yellow. b Detailed insights into the interactions between TTP and the H3-4 subdomains of DSR2. Key interacting residues are shown in stick representation. c In vitro NAD+ cleavage assay using WT or mutants of DSR2 protein. Mutations of the key residues in DSR2-TTP binding interface remarkably reduced NAD+ consumption. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). d Detailed insights into the interactions between TTP and the H3 subdomain of DSR2-B. Key interacting residues in this interface are shown as sticks. e Detailed insights into the interactions between TTP and the H2 subdomain of DSR2. Key residues involved in the hydrophobic interaction between TTP and the H2 subdomain of DSR2 are shown in stick representation. f Close-up view of the interactions between the TTP and the H2-3 subdomains of DSR2. Key interacting residues are shown in stick representation.
Fig. 5
Fig. 5. The molecular basis of DSR2 activation by TTP protein.
a Structural comparison between the DSR2-TTP complex and DSR2-DSAD1 complex. In the structure of the DSR2-DSAD1 complex, the DSR2 and DSAD1 are colored in gray and orange. The color scheme for the DSR2-TTP complex is consistent with that in Fig. 3d. b Close-up view of the inter-domain interaction between the H2 subdomain and SIR2 domain. c In vitro NAD+ degradation assay of WT and mutant DSR2 proteins in the presence or absence of TTP. The NADase activity of DSR2 was self-activated through mutations of key residues in the H2-SIR2 interface. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates). d Compared to the structure of DSR2 in the DSAD1-binding state, the lid region of the SIR2 domain undergoes conformational changes in the TTP-binding state, leading to outward movement and flexibility in the lid region, thus exposing the catalytic pocket (marked with a black circle). e In vitro NAD+ degradation assays investigating the impact of lid region mutations on the NADase activity of DSR2. All experiments were replicated at least three times (mean ± SD, n = 3 independent replicates).
Fig. 6
Fig. 6. Schematic diagram illustrating the NADase activation and inhibition mechanisms of DSR2.
The TTP binds to DSR2 and allosterically activates the SIR2 domain, while the DSAD1 protein encoded by some phages mimics TTP, occupying the same pocket in DSR2 and thereby inhibiting NADase activity.
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