Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics
Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics
复制标题
DOI：
10.1021/jacs.9b10521
复制
发表时间：
2020-01-22
影响因子：
15
通讯作者：
Lisi, George P.
中科院分区：
文献类型：
--
作者：
East, Kyle W.;Newton, Jocelyn C.;Lisi, George P.
CRISPR-Cas9 是一种广泛使用的基因组编辑工具，其功能依赖于 Cas9 核酸内切酶在双链 DNA 中引入位点特异性断裂的能力。在这个系统中，一种有趣的变构通讯被认为可以通过催化 HNH 结构域的灵活性来控制其 DNA 切割活性。在这里，溶液核磁共振实验和新型高斯加速分子动力学 (GaMD) 模拟方法用于捕获 HNH 域内变构信号的结构和动态决定因素。我们揭示了毫秒级动态通路的存在，该通路从与相邻 RuvC 核酸酶连接的区域跨越 HNH，并传播到全长 CRISPR-Cas9 中的 DNA 识别叶。这些发现揭示了 CRISPR-Cas9 HNH 核酸酶内信号转导的潜在途径，增进了我们对激活变构途径的理解。此外，考虑到变构信号在 CRISPR-Cas9 特异性中的作用，这项工作为旨在提高其基因组编辑能力的新型工程工作奠定了机制基础。
CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.