Department of Chemistry, University of Southern California, Los Angeles, California.
Proteins. 2020 Feb;88(2):260-264. doi: 10.1002/prot.25796. Epub 2019 Sep 6.
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 Mg . 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 Mg 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.
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 Cas9 的结构在无配体和底物结合形式下已经可用，但催化活性结构仍不清楚。我们首次尝试探索 Cas9 HNH 结构域的催化机制是基于这样一个合理的假设，即我们正在处理与内切核酸酶 VII 相同的机制，包括假设催化水处于 Mg 的第一壳层。尝试使用 cryo-EM 结构的这种机制迫使我们诱导由 K848（或其他带正电荷的残基）靠近活性位点的运动引起的显著结构变化，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，其中催化水处于 Mg 的第二壳层，并假设 cryo-EM 结构本身就是催化准备结构的合适表示。替代机制表明，如果活性水来自第二壳层，那么与水来自第一壳层的相应屏障相比，计算出的反应势垒较低。
