4.7 Article

Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding

期刊

出版社

ELSEVIER
DOI: 10.1016/j.csbj.2019.12.003

关键词

Polynucleotide phosphorylase (PNPase); PNPase structure; PNPase recognition; RNA oxidation; 8-oxo-7,8-dihydroguanosine; Oxidative stress; MD simulations

资金

  1. Health Effects Institute [4949-RFA14-2/15-3]
  2. Welch Foundation [F-1756]
  3. Artie McFerrin Department of Chemical Engineering at Texas AM
  4. Administrative Department of Science, Technology, and Innovation (COLCIENCIAS) of Colombia
  5. Fulbright [479]
  6. Texas A&M University Graduate Diversity Fellowship

向作者/读者索取更多资源

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.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

4.7
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据