A Tryptophan 'Gate' in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.
A Tryptophan 'Gate' in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.
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DOI：
10.3390/ijms22062848
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发表时间：
2021-03-11
影响因子：
5.6
通讯作者：
Bolt EL
中科院分区：
文献类型：
--
作者：
He L;Matošević ZJ;Mitić D;Markulin D;Killelea T;Matković M;Bertoša B;Ivančić-Baće I;Bolt EL
Cas 3是1类原核CRISPR免疫系统所必需的ssDNA靶向核酸酶-解旋酶，其已用于人类细胞中的基因组编辑。Cas 3-DNA晶体结构表明，ssDNA遵循从解旋酶结构域到HD-核酸酶活性位点的途径，在DNA易位期间需要蛋白质构象灵活性。在遗传研究中，我们注意到，当温度从30 °C升高到37 °C时，Cas 3在CRISPR免疫中的功效急剧降低，这是由未知机制引起的。在这里，使用E.大肠杆菌Cas 3蛋白，我们表明，在较高的温度下降低的核酸酶活性对应于蛋白质结构的可测量的变化。温度对Cas 3的这种影响通过将单个高度保守的色氨酸残基（Trp-406）改变为丙氨酸来减轻。该Cas 3 W 406 A蛋白是一种高活性核酸酶，其功能独立于温度和干扰效应子模块Cascade。Trp-406位于Cas 3 HD和RecA 1结构域的界面，这对于将DNA操纵到核酸酶活性位点中是重要的。基于实验数据的分子动力学模拟显示，温度诱导的Trp-406定位的变化，其阻断或清除ssDNA途径。我们提出Trp-406通过ssDNA进入核酸酶活性位点形成控制Cas 3核酸酶活性的“门”。这些实验中温度的影响可能表明由蛋白质构象变化引起的Cas 3核酸酶活性的变构控制。过度活跃的Cas 3 W 406 A蛋白可以在人类细胞中提供改进的基于Cas 3的基因编辑。
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.
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影响因子：
64.5
作者：
Dillard KE;Brown MW;Johnson NV;Xiao Y;Dolan A;Hernandez E;Dahlhauser SD;Kim Y;Myler LR;Anslyn EV;Ke A;Finkelstein IJ
通讯作者：
Finkelstein IJ
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.
DOI：
10.1126/science.1159689
发表时间：
2008-08-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Brouns SJ;Jore MM;Lundgren M;Westra ER;Slijkhuis RJ;Snijders AP;Dickman MJ;Makarova KS;Koonin EV;van der Oost J
通讯作者：
van der Oost J
影响因子：
16
作者：
Dolan, Adam E.;Hou, Zhonggang;Zhang, Yan
通讯作者：
Zhang, Yan
影响因子：
3.8
作者：
Kekic, Tadija;Fulgosi, Hrvoje;Bertosa, Branimir
通讯作者：
Bertosa, Branimir