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Exploring alternative catalytic mechanisms of the Cas9 HNH domain.
Proteins: Structure, Function, and Bioinformatics ( IF 2.9 ) Pub Date : 2019-09-06 , DOI: 10.1002/prot.25796
Li Na Zhao 1 , Dibyendu Mondal 1 , Arieh Warshel 1
Affiliation  

Understanding the reaction mechanism of CRISPR-associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo- and substrate-bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg2+ . Trying this mechanism with the cryo-EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg2+ and assume that the cryo-EM structure by itself is a suitable representation of a catalytic-ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.

中文翻译:

探索 Cas9 HNH 结构域的替代催化机制。

了解CRISPR相关蛋白9(Cas9)的反应机制对于可编程基因编辑的应用至关重要。尽管可以得到载脂蛋白结合形式和底物结合形式的 Cas9 结构,但其催化活性结构仍不清楚。我们对 Cas9 HNH 结构域催化机制的首次尝试是基于合理的假设,即我们正在处理与核酸内切酶 VII 相同的机制,包括催化水位于 Mg2+ 的第一壳中的假设。在冷冻电镜结构中尝试这种机制迫使我们诱导由靠近活性位点的 K848(或其他带正电荷的残基)移动驱动的显着结构变化,以促进质子转移步骤。在本研究中,我们探索了第二种反应机制,其中催化水位于 Mg2+ 的第二个壳中,并假设冷冻电镜结构本身是催化就绪结构的合适代表。另一种机制表明,如果活性水来自第二壳,则计算出的反应势垒比水来自第一壳时的相应势垒低。
更新日期:2020-01-04
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