Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding
Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding
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DOI：
10.1016/j.csbj.2019.12.003
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发表时间：
2020-01-01
影响因子：
6
通讯作者：
Tamamis, Phanourios
中科院分区：
文献类型：
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作者：
Gonzalez-Rivera, Juan C.;Orr, Asuka A.;Tamamis, Phanourios
RNA的氧化与许多疾病的发展有关。在四种核糖核苷酸中，鸟苷最容易被氧化，从而形成8-氧代-7，8-二氢鸟苷（8-oxoG）。尽管关于细胞如何调节氧化RNA的有害作用的知识有限，但已经开始鉴定参与其调节的细胞因子。这些因素之一是多核苷酸磷酸化酶（PNIPs），一种涉及RNA周转的多功能酶。在本研究中，我们已经研究了PND 4与8-oxoG的原子细节的相互作用，以提供对8-oxoG歧视机制的见解。我们假设，PNIPs亚基合作，形成一个结合位点，使用的PNIPs同源三聚体的中央通道内的动态SFF环。我们使用一种新的方法进化了这个位点，该方法最初从有益突变库中筛选突变体，并使用多纳秒分子动力学模拟评估它们的相互作用。我们发现，进化这个单一位点导致8-oxoG亲和力在1.2和1.5之间和/或选择性在1.5和1.9之间的倍数变化增加。除了8-oxoG结合的改善，K12 Δ pnp与表达突变PNPases的质粒互补引起细胞对H2 O2的耐受性增加。这一观察结果提供了RNA氧化的分子识别和细胞存活之间的明确联系。此外，这项研究提供了一个框架，操纵修饰的RNA蛋白质阅读器，这在合成生物学和epitranscriptomics有潜在的应用。(C)2019年，任作家。由Elsevier B. V.代表计算和结构生物技术研究网络出版。
The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Delta pnp with plasmids expressing mutant PNPases caused increased cell tolerance to H2O2. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics. (C) 2019 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.