Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania.
Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania.
Mol Cell. 2024 Nov 7;84(21):4239-4255.e8. doi: 10.1016/j.molcel.2024.09.002. Epub 2024 Oct 2.
To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.
为了对抗噬菌体感染，III 型 CRISPR-Cas 系统利用环状寡聚腺苷酸（cA）信号来激活各种辅助效应子，包括 CRISPR 相关的 Lon-SAVED 蛋白酶 CalpL，它与反 σ 因子 CalpT 和 ECF 样 σ 因子 CalpS 一起形成一个三分体效应子系统。在这里，我们报告了候选 Cloacimonas acidaminovorans CalpL-CalpT-CalpS 的特性。我们证明 cA 结合触发 CalpL 纤维形成并激活它在 CalpT-CalpS 二聚体中切割 CalpT。这种切割暴露了 CalpT 的 C-降解物，使其被细胞蛋白酶进一步降解。因此，CalpS 被释放出来与 RNA 聚合酶结合，导致大肠杆菌生长停滞。此外，CalpL-CalpT-CalpS 系统受 CalpL 的 SAVED 结构域调节，该结构域是一种环核酶，以连续的三步机制切割 cA。这些发现为 III 型 CRISPR-Cas 免疫信号转导的激活、蛋白水解事件和调控提供了关键的机制细节。
