CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23
CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23
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DOI：
10.1101/2023.11.24.568546
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发表时间：
2023-11
影响因子：
14.9
通讯作者：
S. Grüschow;S. McQuarrie;Katrin Ackermann;Stephen McMahon;B. Bode;T. Gloster;Malcolm F. White
中科院分区：
文献类型：
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作者：
S. Grüschow;S. McQuarrie;Katrin Ackermann;Stephen McMahon;B. Bode;T. Gloster;Malcolm F. White
CRISPR在原核生物中提供适应性免疫。III型CRISPR系统检测入侵的RNA并激活催化Cas 10亚基，该亚基产生一系列核苷酸第二信使以发出感染信号。这些分子结合并激活多种效应蛋白，这些效应蛋白通过降解病毒组分和/或通过干扰细胞代谢的关键方面以减缓病毒复制来提供免疫力。在这里，我们专注于未表征的效应Csx 23，这是广泛存在于霍乱弧菌。当在大肠杆菌中沿着其同源III型CRISPR系统表达时，Csx 23提供针对质粒和噬菌体的免疫力。Csx 23蛋白使用N-末端跨膜α-螺旋结构域定位于膜中，并且具有结合环四腺苷酸（cA 4）的细胞质C-末端结构域，从而激活其防御功能。结构研究揭示了一个四聚体结构与一个新的折叠，结合cA 4特异性。使用脉冲EPR，我们表明，cA 4结合到Csx 23的胞质结构域的跨膜结构域的一个主要的扰动的结果，一致的孔的开放和/或破坏膜的完整性。这项工作揭示了一类新的环核苷酸结合蛋白，并提供了膜相关CRISPR效应器的关键机制细节。
CRISPR provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using a N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.