Structural Determinants of Sleeping Beauty Transposase Activity.
Structural Determinants of Sleeping Beauty Transposase Activity.
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DOI：
10.1038/mt.2016.110
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发表时间：
2016-08
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影响因子：
--
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转座酶是基因组工程中的重要工具，并且对工程化更有效的转座酶有相当大的兴趣。在这里，我们试图了解使用睡美人转座酶决定其活性的因素。最近的工作表明，蛋白质的共同进化的信息可以用来分类组的物理连接，共同进化的残基到元素称为“部门”，这已被证明是有用的理解折叠，变构相互作用，和酶的蛋白质活性。利用大量的突变数据、蛋白质模型和折叠能分析，我们发现：1）睡美人转座酶包含两个区段，它们跨越保守结构域，富含DNA结合残基，表明转座酶的DNA结合功能和核酸内切酶功能是共同进化的; 2）区段残基对突变高度敏感，这些残基的大多数突变强烈地降低转座率; 3）对转座酶的DDE结构域中的折叠自由能具有强影响的突变显著降低转座率。4)影响DNA和蛋白质-蛋白质相互作用的突变通常会降低转座率，尽管大多数高活性突变体也位于蛋白质表面，包括具有蛋白质-蛋白质相互作用的残基。这表明活性亢进是由蛋白质相互作用的修饰引起的，而不是蛋白质折叠的稳定。
Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein co-evolutionary information can be used to classify groups of physically connected, co-evolving residues into elements called ‘sectors’, which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that 1) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; 2) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; 3) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. 4) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.
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