Institute of Structural Biology, Venusberg-Campus 1, University of Bonn, Bonn, Germany.
Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany.
Nucleic Acids Res. 2024 Sep 23;52(17):10520-10532. doi: 10.1093/nar/gkae676.
Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-induced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems.
原核生物 CRISPR-Cas 免疫系统可识别和切割外来核酸。在 III 型 CRISPR-Cas 系统中，激活的识别复合物的 Cas10 亚基合成环寡腺苷酸（cOA），这是激活下游辅助效应蛋白的第二信使。一旦抵御了病毒攻击，消除现存的 cOA 对于限制抗病毒反应和允许细胞恢复至关重要。已经鉴定出各种环核酶家族，它们专门作为独立的酶或作为效应蛋白的结构域来降解 cOA。本文描述了 cA4 激活的 CRISPR Lon 蛋白酶 CalpL 的 SAVED 结构域固有的环核酶活性。我们描述了 cA4 切割的动力学，并鉴定了关键的催化残基。我们证明 cA4 诱导 CalpL 的寡聚化不仅对蛋白酶的激活是必需的，而且对核酶活性也是必需的。此外，CalpL 的核酶活性对蛋白酶反应构成了限制，表明 CalpL/T/S 信号级联的调控机制。这项工作首次证明了催化 SAVED 结构域的存在，并为 CRISPR 防御系统中的转录适应动力学提供了新的见解。
