Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences University of Copenhagen, Blegdamsvej 3-B, Copenhagen, 2200, Denmark.
The Novo Nordisk Foundation Center for Protein Research, Protein Structure & Function Programme, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
Nucleic Acids Res. 2021 Dec 2;49(21):12577-12590. doi: 10.1093/nar/gkab1130.
Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.
III 型 CRISPR-Cas 效应子系统检测外来 RNA，触发 DNA 和 RNA 的切割，并在其 Cas10 亚基中合成环状寡腺苷酸分子 (cA)。cAs 作为第二信使激活辅助核酸酶，导致无差别 RNA 降解，最终导致细胞休眠或死亡。独立的环核酶是 CRISPR 辅助蛋白，通过降解 cA 来下调 III 型系统的强烈免疫反应。这些酶含有一个 CRISPR 相关的 Rossman 折叠 (CARF) 结构域，该结构域结合并切割 cA 分子。在这里，我们展示了来自 Sulfolobus islandicus (Sis) 0811 的独立环核酶在其无配体和催化后状态下的结构。该酶由 N 端 CARF 和 C 端 wHTH 结构域组成。Sis0811 呈现出一种非磷酸二酯水解金属依赖的机制，它可以切割 cA4 环，生成线性腺苷酸产物，从而降低第二信使的水平并关闭细胞抗病毒状态。结构和生化分析揭示了一个螺旋扭转构象变化与关键催化残基的定位之间的耦合，从而以非协同的方式进行 cA4 磷酸二酯水解。
