Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4
Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4
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DOI：
10.1016/j.molcel.2014.09.002
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发表时间：
2014-10-02
期刊：
影响因子：
16
通讯作者：
Conti, Elena
中科院分区：
文献类型：
--
作者：
Benda, Christian;Ebert, Judith;Conti, Elena
Cmr复合物是一种RNA引导的核酸内切酶，作为CRISPR原核防御系统的一部分切割外源RNA靶标。我们使用整合结构生物学方法研究了P. furiosus Cmr复合物的分子结构。我们确定了P. furiosus Cmr 1，Cmr 2，Cmr 4和Cmr 6的晶体结构，并将其与已知的结构信息相结合，以解释复合物的cryo-EM图。为了支持结构测定，我们使用蛋白质交联和质谱法获得了残基特异性相互作用数据。由此产生的伪原子模型揭示了如何超螺旋骨干的复杂的Cmr 4和Cmr 5的聚合原则，以及它是如何在末端由类似的折叠蛋白质的上限。超螺旋的内表面暴露了Cmr 4的保守残基，我们表明这是靶向切割活性所需的。因此，结构和生物化学数据将Cmr 4鉴定为Cmr复合物的保守核糖核酸内切酶。
The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.