Molecular basis for the recognition of phosphorylated STAT1 by importin alpha5.
Molecular basis for the recognition of phosphorylated STAT1 by importin alpha5.
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DOI：
10.1016/j.jmb.2010.07.013
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发表时间：
2010-09-10
影响因子：
5.6
通讯作者：
Cingolani G
中科院分区：
文献类型：
--
作者：
Nardozzi J;Wenta N;Yasuhara N;Vinkemeier U;Cingolani G
干扰素-γ (IFN-γ)刺激会触发转录因子STAT1在701位点的酪氨酸磷酸化，这与从不依赖载体的核胞质穿梭到载体介导的核输入的转换有关。与大多数携带经典核定位信号（cNLS）并与输入蛋白α1结合的底物不同，STAT1具有被输入蛋白α5识别的非经典NLS。在本研究中，我们分析了输入蛋白α5结合磷酸化的STAT1 （pSTAT1）的机制。我们发现pSTAT1的一个同型二聚体被输入α5的一个当量识别，Kd = 191±20 nM。尽管701位点酪氨酸磷酸化对于组装pSTAT1：输入蛋白α5复合体至关重要，但磷酸部分并不是输入蛋白α5的直接决定因素。与经典NLS底物相比，pSTAT1与输入蛋白α5的结合不被n端输入蛋白β结合（IBB）结构域取代，并且需要输入蛋白α5 c端酸性尾部（505-EEDD-508）。输入蛋白α5 ARM重复10的局部展开与pSTAT1具有高亲和力结合。这种展开是由输入蛋白α5的476位的一个保守酪氨酸介导的，该酪氨酸插入Armadillo （ARM）重复的10个螺旋H1-H2-H3之间，从而阻止了分子内螺旋堆积，而这对稳定ARM 10的折叠构象至关重要。在这个位置引入甘氨酸，就像导入α1一样，会破坏与pSTAT1的高亲和力结合，这表明pSTAT1的识别依赖于ARM 10固有的灵活性。利用本文提供的定量化学计量学和结合数据以及文献中的突变信息，我们提出在两个STAT1单体之间输入α5结合，在SH2和dna结合域有两个主要的结合决定因素。体外实验发现，含有两个串联cfosm67启动子的38聚DNA寡核苷酸可以取代pSTAT1的输入α5，这表明DNA可能在细胞核中释放活化的STAT1中发挥作用。
Interferon-γ (IFN-γ) stimulation triggers tyrosine-phosphorylation of transcription factor STAT1 at position 701, which is associated with the switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical Nuclear Localization Signal (cNLS) and bind to importin α1, STAT1 possesses a non-classical NLS recognized by the isoform importin α5. In the present study, we have analyzed the mechanisms by which importin α5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin α5 with Kd = 191 ± 20 nM. Whereas tyrosine-phosphorylation at position 701 is essential to assemble a pSTAT1:importin α5 complex, the phosphate moiety is not a direct binding determinant for importin α5. In contrast to classical NLS substrates, pSTAT1 binding to importin α5 is not displaced by the N-terminal importin β binding (IBB)-domain, and requires the importin α5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin α5 ARM repeat 10 accompanies high affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin α5, which is inserted in between Armadillo (ARM) repeat 10 helices H1-H2-H3, thereby preventing the intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin α1, disrupts high-affinity binding to pSTAT1, suggesting pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this paper together with mutational information available in the literature, we propose importin α5 binds in between two STAT1 monomers, with two major binding determinants in the SH2- and DNA-binding domains. In vitro this model is supported by the observation that a 38mer DNA oligonucleotide containing two tandem cfosM67-promoters can displace importin α5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.6
作者：
Fontes, MRM;Teh, T;Kobe, B
通讯作者：
Kobe, B
影响因子：
4.8
作者：
Fontes, MRM;Teh, T;Kobe, B
通讯作者：
Kobe, B
DOI：
10.1038/7625
发表时间：
1999-04-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Kobe, B
通讯作者：
Kobe, B
影响因子：
4.8
作者：
Koksal, Adem C.;Nardozzi, Jonathan D.;Cingolani, Gino
通讯作者：
Cingolani, Gino