Rational Design of an Artificial Nuclease by Engineering a Hetero-Dinuclear Center of Mg-Heme in Myoglobin
Rational Design of an Artificial Nuclease by Engineering a Hetero-Dinuclear Center of Mg-Heme in Myoglobin
复制标题
通过工程化肌红蛋白中镁血红素的异双核中心合理设计人工核酸酶
DOI：
10.1021/acscatal.0c04572
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发表时间：
2020-12-18
期刊：
影响因子：
12.9
通讯作者：
Lin, Ying-Wu
中科院分区：
文献类型：
--
作者：
Luo, Jie;Du, Ke-Jie;Lin, Ying-Wu
人工核酸酶的设计在生物技术和生物医学中是必不可少的，但很少有人工核酸酶能同时切割和降解DNA分子。血红素蛋白是DNA裂解的潜在酶。使用一种小的血红素蛋白，肌红蛋白(Mb)作为模型蛋白，我们在血红素远端位点设计了一个金属结合基序[1-His-1-Glu](天然的His64和突变的Glu29)。L29E Mb的X射线晶体结构表明，L29E Mb的单个突变体不仅能够有效地切割DNA，而且能够在镁离子与血红素远端结合的情况下降解DNA。蛋白质-DNA复合体的分子对接显示了它们界面上的多个氢键相互作用，涉及DNA的细小和主要沟槽。此外，末端的Arg45和配体Glu29都被鉴定为核酸酶活性的关键残基。本研究报道了镁-血红素水桥异双核中心(Mg2+-H2O-Fe3+)的结构，其功能与天然核酸酶中的同核中心(MgA(2+)-H2O-MgB2+)相似，表明该Mb络合物是一种有效的人工核酸酶。
Design of artificial nucleases is essential in biotechnology and biomedicine, whereas few artificial nucleases can both cleave and degrade DNA molecules. Heme proteins are potential enzymes for DNA cleavage. Using a small heme protein, myoglobin (Mb), as a model protein, we engineered a metal-binding motif of [1-His-1-Glu] (native His64 and mutated Glu29) in the heme distal site. The single mutant of L29E Mb was capable of not only efficient DNA cleavage but also DNA degradation upon Mg2+ binding to the heme distal site, as shown by an X-ray crystal structure of the Mg2+-L29E Mb complex. Molecular docking of the protein-DNA complex revealed multiple hydrogen-bonding interactions at their interfaces, involving both minor and major grooves of DNA. Moreover, both the distal Arg45 and the ligand Glu29 were identified as critical residues for the nuclease activity. This study reports the structure of a water-bridged heterodinuclear center of Mg-heme (Mg2+-H2O-Fe3+), showing a similar function as the homodinuclear center (MgA(2+)-H2O-MgB2+) in natural nuclease, which indicates that the Mg2+-L29E Mb complex is an effective artificial nuclease.