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Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal

机译：多重RBD蛋白致死性RNA结合界面的化学位移图谱

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The Drosophila protein Sex-lethal (Sxl) contains two RNP consensus-type RNA-binding domains (RBDs) separated by a short linker sequence. Both domains are essential for high-affinity binding to the single-stranded polypyrimidine tract (PPT) within the regulated 3' splice site of the transformer (tra) pre-mRNA. In this paper, the effect of RNA binding to a protein fragment containing both RBDs from Sxl (Sxl-RBD1 + 2) has been characterized by heteronuclear NMR. Nearly complete (85-90%) backbone resonance assignments have been obtained for unbound and RNA-bound states of Sxl-RBD1 + 2. A comparison of amide 1H and 15N chemical shifts between free and bound states has highlighted residues which respond to RNA binding. The beta-sheets in both RBDs (RBD1 and RBD2) form an RNA interaction surface, as has been observed in other RBDs. A significant number of residues display different behavior when comparing RBD1 and RBD2. This argues for a model in which RBD1 and RBD2 of Sxl have different or nonanalogous points of interaction with the tra PPT. R142 (in RBD2) exhibits the largest chemical shift change upon RNA binding. The role of R142 in RNA binding was tested by measuring the Kd of a mutant of Sxl-RBD1 + 2 in which R142 was replaced by alanine. This mutant lost the ability to bind RNA, showing a correlation with the chemical shift difference data. The RNA-binding affinities of two other mutants, F146A and T138I, were also shown to correlate with the NMR observations.
机译：果蝇蛋白性致死（Sxl）包含两个RNP共有型RNA结合结构域（RBD），由短接头序列分隔。这两个域对于在变压器（tra）pre-mRNA的调控3'剪接位点内与单链聚嘧啶束（PPT）的高亲和力结合都是必不可少的。在本文中，RNA结合了包含来自Sxl的两个RBD（Sx1-RBD1 + 2）的RBD的蛋白质片段的作用已通过异核NMR进行了表征。对于Sxl-RBD1 + 2的未结合状态和RNA结合状态，已经获得了几乎完整的（85-90％）骨架共振分配。比较游离态和结合态之间酰胺1H和15N的化学位移，突出了对RNA结合有响应的残基。正如在其他RBD中所观察到的那样，两个RBD（RBD1和RBD2）中的β-折叠均形成RNA相互作用表面。比较RBD1和RBD2时，大量残基表现出不同的行为。这证明了一个模型，其中Sxl的RBD1和RBD2与tra PPT有不同或非相似的相互作用点。 R142（在RBD2中）在RNA结合后表现出最大的化学位移变化。通过测量其中R142被丙氨酸替代的Sx1-RBD1 + 2突变体的Kd来测试R142在RNA结合中的作用。该突变体失去了结合RNA的能力，显示出与化学位移差异数据的相关性。还显示了另外两个突变体F146A和T138I的RNA结合亲和力与NMR观察结果相关。

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