In the long-term and intense survival competition between bacteria, archaea, and bacteriophages, they have evolved various complex antiviral systems to resist the invasion of bacteriophages. The latest research reveals that approximately 7% of sequenced bacterial genomes have at least one defense system, among which the depletion of nicotinamide adenine dinucleotide (NAD+) is a common strategy to combat phage induced miscarriage infections. The hydrolysis of NAD+is mainly performed by proteins containing TIR and Sir2 domains.

Recently, Professor Ouyang Songying's team from the School of Life Sciences at Fujian Normal University collaborated to publish a paper titled "Mechanical Basis for the Allosteric Activation of NADase Activity in the Sir2 HerA Antiphage Defense System" online in Nature Communications. This study uses bioinformatics, structural biology, and experimental techniques such as biochemistry to reveal the molecular mechanism of how the Sir2 HerA system is activated and exerts its anti phage function in pathogen immunity.The Sir2 protein family is a conserved protein family widely present in bacteria, archaea, and higher eukaryotes. In eukaryotic organisms, Sir2 protein utilizes NAD+as a cofactor to function as a protein deacetylase or ADP ribosyltransferase. However, multiple anti phage defense systems in prokaryotes, such as the SPARSA system previously published by Ouyang Songying's team (Nat Communi 10.1038/s41467-023-44660-7) and Sir2 HerA, both contain Sir2 domain proteins. Although multiple Sir2 related anti phage systems have been identified recently, it is still unclear how Sir2's activity as an NAD+hydrolase is activated. In this study, researchers selected the Sir2 HerA system in the Staphylococcus aureus strain as the research object. Firstly, Pull Down experiments were conducted to discover the interaction between Sir2 and HerA. In vitro experiments showed that Sir2 alone did not have NAD+enzyme activity, but after forming a complex with HerA, the NAD+hydrolase activity of Sir2 protein was activated, demonstrating strong ability to hydrolyze NAD+. However, in the absence of phage infection, the enzymatic activity of Sir2 is inhibited by the physiological concentration of ATP within the cell.

To explain how HerA activates the NAD+hydrolase activity of Sir2, researchers further used cryo electron microscopy (Cryo EM) to analyze the structure of Sir2 HerA complex binding to ADPR (resolution of 2.81 Å). This complex is a super complex composed of 12 Sir2 molecules and 6 HerA molecules. By analyzing the structure of the complex, researchers found that Sir2 protein is arranged in a two-layer hexagonal ring structure, but only the density of NAD+hydrolysis product ADPR was observed in the Sir2 hexagonal ring in direct contact with HerA protein. ADPR is located between the small domain and Rossmann domain of Sir2 protein, which is a typical site for Sir2 protein to bind with NAD+. However, ADPR was not found in the Sir2 molecule in the other layer of hexagonal ring.

                                                                                                      Figure 1 Cryo electron microscopy structure of Sir2 HerA and the structural basis of Sir2 activation by HerA binding

In order to investigate the activation mechanism of Sir2 enzyme activity, researchers compared the Sir2 structure that binds to ADPR with the Sir2 structure that does not bind to ADPR in the lower layer, as well as the Sir2 model predicted by AlphaFold. The results showed that the α 15 helix above the Sir2 enzyme active site in direct contact with HerA became a loop and bent away from ADPR, which increased the Sir2 NAD+enzyme activity pocket. Researchers speculate that this conformational change may allow NAD+to approach the enzyme active center of Sir2 and be hydrolyzed. To verify the above hypothesis, researchers used BLI technology to discover that Sir2 alone does not bind to NAD+, while the Sir2 HerA mutant has the ability to bind to NAD+. In addition, by removing the α 15 helix of Sir2 monomer and conducting in vitro NAD+enzyme activity experiments, it was found that the truncated Sir2 protein monomer can exhibit NAD+enzyme activity without the binding of HerA. These results indicate that the α 15 helix plays an important role in activating the Sir2 HerA system.

In summary, researchers have proposed a Sir2 HerA system complex assembly and allosteric activation model, which provides a detailed explanation of the molecular mechanism of the Sir2 HerA system in resisting phage invasion. These findings not only deepen our understanding of the Sir2 HerA defense system in prokaryotes, but also provide a theoretical basis for further research on the immune system of microorganisms against bacteriophages.                                                                                                                               Figure 2 Summary of the molecular mechanism of Sir2 HerA against bacteriophages

Ouyang Songying, Professor of School of Life Sciences, Fujian Normal University, and Southern Biomedical Research Center, and Xiong Xiaoli and He Jun, Professors of Chinese Academy of Sciences, Guangzhou Institute of Biomedicine and Health, are the co corresponding authors of this article. Associate researcher Zhen Xiangkai, School of Life Sciences, Fujian Normal University, Dr. Liu Zihe and Dr. Wang Xurong, as well as Dr. Zhou Biao (Guangzhou National Laboratory) and Dr. Zhao Heyu of the Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health, are the co first authors of this article.