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首页> 外文期刊>Journal of Molecular Biology >The x-ray crystal structure of the first RNA recognition motif and site-directed mutagenesis suggest a possible HuR redox sensing mechanism.
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The x-ray crystal structure of the first RNA recognition motif and site-directed mutagenesis suggest a possible HuR redox sensing mechanism.

机译：第一个RNA识别基序的X射线晶体结构和定点诱变表明可能存在HuR氧化还原感应机制。

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摘要

Hu-antigen R (HuR) is a ubiquitous RNA-binding protein that comprises three RNA recognition motifs (RRMs). The first two tandem RRMs are known to bind to AU-rich elements (AREs) in the 3'-untranslated region of many mRNAs. The third RRM is connected to the second RRM through a basic hinge region that contains a localization signal termed HuR nucleocytoplasmic shuttling. Binding of HuR to the ARE in the 3'-untranslated region of mRNA leads to nuclear export, stabilization, and/or translational de-repression of the mRNA, resulting in upregulation of the encoded protein. Among the various ARE binding proteins known to date, HuR is still the only known ubiquitous antagonist of posttranscriptional gene silencing by AREs. Given the wide repertoire of known and suspected targets of HuR, it is considered to be a central node in the ARE pathway. Here, the x-ray crystal structure of the first RRM of HuR (amino acids 18-99) at 2.0 A resolution is presented. The overall fold consists of two alpha-helices and a four-stranded beta-sheet, with a beta1-alpha1-beta2-beta3-alpha2-beta4 topology and a beta-hairpin between alpha2 and beta4. The asymmetric unit consists of four chains. The large crystal contact interfaces observed between chains A/B and C/D contain hydrophobic residues located at the alpha-helix side of the fold, opposite to the RNA-binding interface. This hydrophobic region structurally resembles the protein-protein interaction site of RRM domains of other proteins. Because the nature of the assumed HuR homodimerization is mechanistically not well understood to date, we used site-directed mutagenesis, analytical size-exclusion chromatography and multiangle light scattering to investigate HuR interactions via the RRM hydrophobic region. Our data indicate that in vitro, HuR RRM1 and RRM1,2 homodimerization involves a disulfide bond at cysteine 13. This homodimerization mode may have a functional significance in redox modulation of HuR activity in response to oxidative stress. Because HuR is involved in many diseases (e.g., cancer, cachexia, and inflammatory bowel disease), the presented structure may provide a basis for rational drug design.
机译：Hu抗原R（HuR）是一种普遍存在的RNA结合蛋白，包含三个RNA识别基序（RRM）。已知前两个串联RRM与许多mRNA的3'非翻译区中的富AU元件（ARE）结合。第三RRM通过基本铰链区连接到第二RRM，所述基本铰链区包含称为HuR核质穿梭的定位信号。 HuR与mRNA 3'-非翻译区中的ARE结合导致mRNA的核输出，稳定和/或翻译抑制，导致编码蛋白的上调。在迄今为止已知的各种ARE结合蛋白中，HuR仍然是ARE已知的转录后基因沉默的唯一普遍存在的拮抗剂。鉴于已知的和可疑的HuR靶标种类繁多，它被认为是ARE途径中的中心节点。在此，给出了HuR的第一个RRM（氨基酸18-99）在2.0 A分辨率下的X射线晶体结构。整体折叠由两个alpha螺旋和一个四链的beta折叠组成，具有beta1-alpha1-beta2-beta3-alpha2-beta4拓扑结构和位于alpha2和beta4之间的beta发夹。不对称单元由四个链组成。在A / B和C / D链之间观察到的大晶体接触界面含有疏水残基，位于残基的α-螺旋侧，与RNA结合界面相反。该疏水区在结构上类似于其他蛋白质的RRM结构域的蛋白质-蛋白质相互作用位点。由于迄今为止尚未从机理上很好地理解假定的HuR同二聚体的性质，因此我们使用了定点诱变，分析尺寸排阻色谱和多角度光散射来研究经由RRM疏水区的HuR相互作用。我们的数据表明，在体外，HuR RRM1和RRM1,2同型二聚化涉及半胱氨酸13上的二硫键。该同型二聚化模式在响应氧化应激的HuR活性的氧化还原调节中可能具有功能性意义。由于HuR涉及许多疾病（例如，癌症，恶病质和炎症性肠病），因此所提出的结构可能为合理药物设计提供基础。

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