Exploring alternative catalytic mechanisms of the Cas9 HNH domain
Exploring alternative catalytic mechanisms of the Cas9 HNH domain
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DOI：
10.1002/prot.25796
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发表时间：
2020-02
期刊：
影响因子：
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通讯作者：
Li na Zhao;D. Mondal;A. Warshel
中科院分区：
文献类型：
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作者：
Li na Zhao;D. Mondal;A. Warshel
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的结构以载脂蛋白和底物结合形式存在，但催化活性结构仍不清楚。我们探索Cas9 HNH结构域的催化机制的第一次尝试是基于我们处理与内切核酸酶VII相同的机制的合理假设，包括催化水在Mg 2+的第一壳中的假设。尝试使用cryo-EM结构的这种机制迫使我们通过K848（或其他带正电荷的残基）靠近活性位点的移动来诱导显著的结构变化，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Mg 2+的第二层壳中，并假设cryo-EM结构本身是催化就绪结构的合适代表。另一种机理表明，如果活性水来自第二壳层，则计算的反应势垒低于水来自第一壳层时的相应势垒。
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.