Coupling distinct enzymatic effectors emerges as an efficient strategy for defense against phage infection in bacterial immune responses, such as the widely studied nuclease and cyclase activities in the type III CRISPR-Cas system. However, concerted enzymatic activities in other bacterial defense systems are poorly understood. Here, we biochemically and structurally characterize a two-component defense system DUF4297–HerA, demonstrating that DUF4297–HerA confers resistance against phage infection by cooperatively cleaving dsDNA and hydrolyzing ATP. DUF4297 alone forms a dimer, and HerA alone exists as a nonplanar split spiral hexamer, both of which exhibit extremely low enzymatic activity. Interestingly, DUF4297 and HerA assemble into an approximately 1 MDa supramolecular complex, where two layers of DUF4297 (6 DUF4297 molecules per layer) linked via inter-layer dimerization of neighboring DUF4297 molecules are stacked on top of the HerA hexamer. Importantly, the complex assembly promotes dimerization of DUF4297 molecules in the upper layer and enables a transition of HerA from a nonplanar hexamer to a planar hexamer, thus activating their respective enzymatic activities to abrogate phage infection. Together, our findings not only characterize a novel dual-enzyme anti-phage defense system, but also reveal a unique activation mechanism by cooperative complex assembly in bacterial immunity.

耦合不同的酶效应子已成为防御细菌免疫反应中噬菌体感染的有效策略，例如 III 型 CRISPR-Cas 系统中广泛研究的核酸酶和环化酶活性。然而，人们对其他细菌防御系统中协同酶活性知之甚少。在这里，我们从生化和结构上表征了双组分防御系统 DUF4297-HerA，证明 DUF4297-HerA 通过协同切割 dsDNA 和水解 ATP 赋予对噬菌体感染的抵抗力。 DUF4297 单独形成二聚体，而 HerA 单独作为非平面分裂螺旋六聚体存在，两者都表现出极低的酶活性。有趣的是，DUF4297 和 HerA 组装成大约 1 MDa 的超分子复合物，其中两层 DUF4297（每层 6 个 DUF4297 分子）通过相邻 DUF4297 分子的层间二聚化连接，堆叠在 HerA 六聚体的顶部。重要的是，复杂的组装促进了上层 DUF4297 分子的二聚化，并使 HerA 从非平面六聚体转变为平面六聚体，从而激活它们各自的酶活性以消除噬菌体感染。总之，我们的研究结果不仅表征了一种新型双酶抗噬菌体防御系统，而且揭示了细菌免疫中协同复合物组装的独特激活机制。