Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain
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DOI：
10.7554/elife.46500
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发表时间：
2019-07
期刊：
影响因子：
7.7
通讯作者：
Zhicheng Zuo;A. Zolekar;Kesavan Babu;Victor. J. T. Lin;Hamed S. Hayatshahi;R. Rajan;Yu-Chieh Wang;Jin Liu
中科院分区：
文献类型：
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作者：
Zhicheng Zuo;A. Zolekar;Kesavan Babu;Victor. J. T. Lin;Hamed S. Hayatshahi;R. Rajan;Yu-Chieh Wang;Jin Liu
来自化脓性链球菌的CRISPR相关核酸内切酶Cas9（SpyCas 9）与可编程的单向导RNA（sgRNA）一起沿着，已被用作重要的基因组编辑工具。尽管最近在确定SpyCas 9结构和DNA切割机制方面取得了进展，SpyCas 9的催化HNH核酸酶结构域的切割能力构象在很大程度上仍然是难以捉摸和有争议的。通过整合计算和实验方法，我们揭示并验证了活化的Cas9-sgRNA-DNA三元复合物，其中HNH结构域整齐地准备切割靶DNA链。在该催化模型中，HNH采用D839-H840-N863的催化三元组用于裂解催化，而不是先前涉及的D839-H840-D861、D837-D839-H840或D839-H840-D861-N863。我们的研究为定义HNH结构域的催化构象提供了关键信息，并推进了关于Cas9介导的DNA切割的构象激活的知识。
The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.