Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Oct 27;15(1):9269.
doi: 10.1038/s41467-024-53614-6.

Mechanistic basis for the allosteric activation of NADase activity in the Sir2-HerA antiphage defense system

Affiliations

Mechanistic basis for the allosteric activation of NADase activity in the Sir2-HerA antiphage defense system

Xiangkai Zhen et al. Nat Commun. .

Abstract

Sir2-HerA is a widely distributed antiphage system composed of a RecA-like ATPase (HerA) and an effector with potential NADase activity (Sir2). Sir2-HerA is believed to provide defense against phage infection in Sir2-dependent NAD+ depletion to arrest the growth of infected cells. However, the detailed mechanism underlying its antiphage activity remains largely unknown. Here, we report functional investigations of Sir2-HerA from Staphylococcus aureus (SaSir2-HerA), unveiling that the NADase function of SaSir2 can be allosterically activated by the binding of SaHerA, which then assembles into a supramolecular complex with NADase activity. By combining the cryo-EM structure of SaSir2-HerA in complex with the NAD+ cleavage product, it is surprisingly observed that Sir2 protomers that interact with HerA are in the activated state, which is due to the opening of the α15-helix covering the active site, allowing NAD+ to access the catalytic pocket for hydrolysis. In brief, our study provides a comprehensive view of an allosteric activation mechanism for Sir2 NADase activity in the Sir2-HerA immune system.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Sir2 alone in SaSir2-HerA does not exhibit NADase activity, which can be activated upon the binding of HerA.
a Schematic diagram of the Sir2 and HerA operon of S. aureus. b In vitro NADase activity of SaSir2 and SaSir2-HerA; SaSir2 could not hydrolyze NAD+; in contrast, a mixture of SaSir2 and SaHerA exhibited NADase activity. c The gel filtration profile and SDS‒PAGE results of copurified SaSir2-HerA. d AUC result for apo SaSir2, suggesting that it behaved as a homodimer in solution. e Effects of different concentrations of ATP on the NADase activity of SaSir2-HerA. Data were presented as mean ± SD in histograms; n = 3 independent experiments. P-value determined by paired two-tailed t-tests is indicated in the figure. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Cryo-EM structure of the homotrimeric SaHerA.
a Cryo-EM density maps of the trimeric SaHerA from different views. b Ribbon diagram of the structure of the homotrimeric SaHerA from the same views as in (a). c Structure of SaHerA generated by AlphaFold; the HAS domain, ATPase domain and helical bundle are colored orange, cyan and blue, respectively. d The portions discernible in the apo SaHerA structure are boxed. e, f Detailed interactions among the protomers mediated by the helical-bundle domain of SaHerA.
Fig. 3
Fig. 3. Cryo-EM structure of the SaSir2-HerA supramolecular complex.
a Different views of the cryo-EM density map of the SaSir2-HerA complex, SaSir2 and SaHerA are shown with the protomers color coded. b Atomic model built into the cryo-ME map, which is colored as (a). c, d Cartoon mode and surface mode of the N-terminal domain of SaHerA. e Top view of the SaHerA hexamer; the two clefts between the two trimers are illustrated.
Fig. 4
Fig. 4. Structural analysis of SaSir2 domain and the assembly of the two-layered SaSir2 dodecamer.
a SaSir2 exhibits a typical Rossmann fold. b The closest structurally similar SaSir2 NADase (EcSir2, PDB 8SXX) to SaSir2. c Overlay of SaSir2 and EcSir2 highlighting the differences among SaSir2, EcSir2 and other Sir2 domain-containing proteins. The additional loop and α helix bundle are highlighted in blue. d Cartoon model of the SaSir2 dodecamer. e The hexameric layer of SaSir2 protomers that interact directly with SaHerA (upper layer). f The bottom layer of SaSir2 protomers that do not interact with SaHerA. g Homodimer units of SaSir2. h-i Enlarged view of the interactions shown in (g). j Neighboring SaSir2 dimer unit were pulled together by Sir2 molecules in the bottom layer (Molecule B) and the top layer (Molecule C). k Enlarged view of the β-hairpin formed in (j). l Detailed interactions of the interfaces shown in (j).
Fig. 5
Fig. 5. Interactions between SaSir2 and SaHerA.
a Surface and cartoon representation of SaSir2-HerA, showing the minimal SaSir2-HerA unit. b, c Specific interactions between SaSir2 and SaHerA; the residues involved in their interactions are shown as sticks. d Deletion of the N-terminal residues of Sir2 and HerA, encompassing residues 1–34 and 1–35, simultaneously abolished the NADase activity of Sir2-HerA. Data were presented as mean ± SD in histograms from three replicates. P-values were determined by paired two-tailed t-tests. e In vitro pull-down assays of SaSir2 with an N-terminal 1–34 deletion and HerA with an N-terminal 1–35 deletion, His-tagged HerA was used as bait, Sir2 was used as a pray. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Mechanism of NAD+ catalysis of SaSir2 NADase.
a Cut-open sliced view of the NAD+ binding pocket of SaSir2top, the NAD+ cleavage product is shown in sticks. b An enlarged view of ADPR coordination by the key residues of SaSir2. c Effects of mutations on the ADPR-coordinated residues in the in vitro NADase assays. Data were presented as mean ± SD in histograms from three replicates. P-values were determined by paired two-tailed t-tests. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. The allosteric activation mechanism of SaSir2 NADase activity.
a Overlay of SaSir2 in the bottom layer (ADPR-free) and the upper layer (ADPR-bound) in cartoon representation. The SaSir2 molecules in the bottom and upper layers are colored white and cyan, respectively. α15 (residues 316-325) is colored blue, and the corresponding loop is highlighted in red. b Structural alignment of SaSir2top docked into NAD+ and SaSir2bottom (left) and enlarged view of the steric clashes between the NAD+ and SaSir2bottom, the NAD+ is shown in dotted surface mode (right). c, d BLI sensorgrams of SaSir2, SaSir2-HerA and NAD+. From the top curve to the bottom curve, NAD+ was subjected to twofold serial dilutions starting at 16.5 µM. e In vitro NADase activity of wild-type Sir2 and Sir2 with α15 deletion, as similar to the complex of SaSir2-HerA, the NAD+ level in SaSir2 with α15 deletion is significant lower that the wild-type SaSir2. Data were presented as mean ± SD in histograms from three replicates. Unpaired two-tailed Student’s tests were performed. f The proposed mechanism of the antiphage function of SaSir2-HerA. Source data are provided as a Source Data file.

References

    1. Georjon, H. & Bernheim, A. The highly diverse antiphage defence systems of bacteria. Nat. Rev. Microbiol. 21, 686–700 (2023). - PubMed
    1. Watson, B. N. J., Steens, J. A., Staals, R. H. J., Westra, E. R. & van Houte, S. Coevolution between bacterial CRISPR-Cas systems and their bacteriophages. Cell Host Microbe29, 715–725 (2021). - PubMed
    1. Hille, F. et al. The biology of CRISPR-Cas: backward and forward. Cell172, 1239–1259 (2018). - PubMed
    1. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science359, eaar4120 (2018). - PMC - PubMed
    1. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science369, 1077–1084 (2020). - PMC - PubMed

Publication types

LinkOut - more resources