pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability
pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability
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DOI：
10.1002/pro.2243
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发表时间：
2013-05-01
期刊：
影响因子：
8
通讯作者：
Brooks, Charles L., III
中科院分区：
文献类型：
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作者：
Law, Sean M.;Zhang, Bin W.;Brooks, Charles L., III
Tombusviruses，如Carnation Italian ringspot virus（CIRV），编码一种称为p19的蛋白质同源二聚体，能够通过结合并隔离短干扰RNA（siRNA）远离RNA沉默途径来抑制感染宿主中的RNA沉默。P19结合稳定性已被证明是敏感的pH值的变化，但涉及的具体氨基酸残基仍不清楚。使用恒定pH分子动力学模拟，我们已经确定了关键的pH依赖性残基，影响CIRV p19 siRNA结合稳定性在不同的pH范围内的基础上计算的变化，从每个可滴定的残基的自由能贡献。在高pH下，Lys 60、Lys 67、Lys 71和Cys 134的去质子化对结合稳定性的影响最大。类似地，几个酸性残基（Asp 9、Glu 12、Asp 20、Glu 35和/或Glu 41）在低pH下的去质子化导致结合稳定性降低。在中性pH下，残基Glu 17和His 132提供了结合稳定性的小幅增加，并且我们发现siRNA结合的最佳pH范围在7.0和10.0之间。总的来说，我们的研究结果进一步通知最近的实验，并在良好的协议与pH值依赖性的结合曲线的数据。
Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile.