Computational redesign of endonuclease DNA binding and cleavage specificity
Computational redesign of endonuclease DNA binding and cleavage specificity
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DOI：
10.1038/nature04818
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发表时间：
2006-06-01
期刊：
影响因子：
64.8
通讯作者：
Baker, David
中科院分区：
文献类型：
--
作者：
Ashworth, Justin;Havranek, James J.;Baker, David
DNA结合特异性的重编程是计算蛋白设计的一个重要挑战，它测试了当前对蛋白质- DNA识别的理解，并且对生物技术和医学具有相当大的实际意义（1-6）。在这里，我们描述了利用物理上真实的原子水平力场对内含子编码的归巢内切酶I-MsoI(7)的切割特异性的计算重新设计（8,9）。使用硅屏，我们确定了预计会破坏野生型酶结合的单碱基对取代，然后使用蒙特卡罗采样优化了这些不利取代周围氨基酸簇的身份和构象（10）。通过实验表征了一种重新设计的酶，该酶预计会显示改变的靶点特异性，同时保持野生型的结合亲和力。重新设计的酶结合和切割重新设计的识别位点的效率是野生型酶的1万倍，其目标识别水平与原始内切酶相当。重新设计的核酸酶识别位点复合物的x射线晶体学结构证实了计算预测界面的准确性。这些结果表明，计算蛋白设计方法可以在创建用于基因治疗和其他应用的新型高特异性内切酶方面发挥重要作用。
The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein - DNA recognition, and has considerable practical relevance for biotechnology and medicine(1-6). Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI(7) using a physically realistic atomic-level forcefield(8,9). Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling(10). A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site similar to 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.