Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB.
Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB.
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对核糖核酸酶 III 和调节性大结构域蛋白 YmdB 的复合物进行计算和实验结合分析。
DOI：
10.1002/prot.24751
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Nicholson,AllenW
中科院分区：
文献类型：
--
作者：
Paudyal,Samridhdi;Alfonso-Prieto,Mercedes;Carnevale,Vincenzo;Redhu,ShivK;Klein,MichaelL;Nicholson,AllenW
核糖核酸酶 III 是一种保守的细菌核酸内切酶，可切割多种编码和非编码 RNA 中的双链 (ds) 结构。 RNase III 受到多层次的控制，进而赋予全局转录后调控。大肠杆菌大结构域蛋白YmdB直接与RNase III相互作用，并且体内YmdB量的增加与RNase III活性的降低相关。在这里，进行了基于计算的结构分析，以确定 YmdB-RNase III 相互作用的原子级特征。单体E的对接。 coliYmdB 与 E 的同源模型。 coliRNase III 同二聚体产生一种复合物，该复合物表现出保守的 YmdB 残基 R40 与亚基界面处的特定 RNase III 残基的相互作用。表面等离子共振 (SPR) 分析得出复合物的 KD 为 61 nM，对应于 -9.9 kcal/mol 的结合自由能 (ΔG)。 YmdB R40 和 RNase III D128 通过硅丙氨酸诱变被鉴定为热力学上重要的相互作用伙伴。与预测一致，通过 SPR 测量，YmdB R40A 突变导致 KD 增加 16 倍（ΔΔG= +1.8 kcal/mol），两个 RNase III 亚基（D128A/D128′A）中的 D128A 突变导致 KD 增加 83 倍（ΔΔG= +2.7 kcal/mol）。 CD 光谱显示，D128A/D128'A 突变的更大影响可能反映了 RNase III 二级结构的改变，这也可以解释体外催化活性的显着降低。讨论了与潜在 RNase III 调控机制相关的模型复合物的特征。蛋白质 2015; 83：459-472。 © 2014 作者。蛋白质：结构、功能和生物信息学 由 Wiley periodicals, Inc. 出版
Ribonuclease III is a conserved bacterial endonuclease that cleaves double‐stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post‐transcriptional regulation. TheEscherichia colimacrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amountin vivocorrelates with a reduction in RNase III activity. Here, a computational‐based structural analysis was performed to identify atomic‐level features of the YmdB‐RNase III interaction. The docking of monomericE. coliYmdB with a homology model of theE. coliRNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided aKDof 61 nMfor the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified byin silicoalanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16‐fold increase inKD(ΔΔG= +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128′A) causes an 83‐fold increase inKD(ΔΔG= +2.7 kcal/mol). The greater effect of the D128A/D128′A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activityin vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.