Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.
Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.
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DOI：
10.1063/5.0128815
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发表时间：
2022-12
期刊：
影响因子：
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通讯作者：
Helen B. Belato;C. Norbrun;Jinping Luo;Chinmai Pindi;Souvik Sinha;Alexandra M. D’Ordine;G. Jogl;G. Palermo;George P. Lisi
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文献类型：
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作者：
Helen B. Belato;C. Norbrun;Jinping Luo;Chinmai Pindi;Souvik Sinha;Alexandra M. D’Ordine;G. Jogl;G. Palermo;George P. Lisi
多结构域蛋白中的变构信号是空间上遥远的功能位点之间通信的驱动因素。了解大分子多域蛋白质的变构偶联机制是实现系统时空调控的最有前途的途径。最近CRISPR-Cas9在分子生物学和医学上的应用的爆炸性增长使得人们有必要了解Cas9的原子水平蛋白质动力学是如何影响其生物物理特性的，这些动力学是其变构串扰的驱动力。在这项研究中，我们使用核磁共振(核磁共振)和计算的协同方法来精确定位耐热GeoCas9的HNH结构域中的变构热点。我们发现K597突变为丙氨酸破坏了盐桥网络，进而改变了结构、变构运动的时间尺度和GeoHNH结构域的热稳定性。在被广泛研究的中嗜性化脓性链球菌Cas9中，这种从赖氨酸到丙氨酸的同源突变类似地改变了SpHNH结构域的动态。我们先前已经证明，通过突变引起的变构改变是SpCas9(ESpCas9)特异性增强的一个来源。因此，在GeoCas9中可能也是如此。
Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.