Mutations in DNA-binding loop of NFAT5 transcription factor produce unique outcomes on protein-DNA binding and dynamics.
Mutations in DNA-binding loop of NFAT5 transcription factor produce unique outcomes on protein-DNA binding and dynamics.
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DOI：
10.1021/jp403310a
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发表时间：
2013-10-24
期刊：
影响因子：
--
通讯作者：
Panchenko AR
中科院分区：
文献类型：
--
作者：
Li M;Shoemaker BA;Thangudu RR;Ferraris JD;Burg MB;Panchenko AR
活化 T 细胞核因子 5（NFAT5 或 TonEBP）是 Rel 家族转录激活因子，在高渗条件下被激活。多项研究指出核易位和 DNA 结合之间可能存在联系。然而，NFAT5核转位的机制以及DNA结合对将NFAT5保留在细胞核中的影响尚不清楚。最近的实验表明，DNA 结合环和二聚化界面中引入的不同突变对于 DNA 结合很重要，其中一些突变降低了 NFAT5 的核质比。为了了解这些突变的机制，我们模拟了它们对蛋白质动力学和 DNA 结合的影响。我们表明，不含 DNA 的 NFAT5 复合物比含 DNA 的复合物灵活得多。此外，DNA 结合显着稳定了整个二聚体复合物，而 NFAT5 二聚体在没有 DNA 的情况下仅具有一定程度的稳定性。发现来自同一 DNA 结合环的两组 NFAT5 突变具有不同的 DNA 特异性和非特异性结合机制。 R217A/E223A/R226A（R293A/E299A/R302A，使用异构体 c 编号）突变体的特点是与 DNA 的结合显着受损，且复合体灵活性更高。相反，T222D（异构体 c 中的 T298D）突变是一种潜在的拟磷突变，使整体复合物更加刚性，并且不会显着影响 DNA 结合。因此，NFAT5 核质比的降低可归因于三重突变体与 DNA 的结合减少，而 T222D 突变体则表明还有其他机制在起作用。
The nuclear factor of activated T cells 5 (NFAT5 or TonEBP) is a Rel family transcriptional activator and is activated by hypertonic conditions. Several studies point to a possible connection between nuclear translocation and DNA binding; however, the mechanism of NFAT5 nuclear translocation and the effect of DNA binding on retaining NFAT5 in the nucleus are largely unknown. Recent experiments showed that different mutations introduced in the DNA-binding loop and dimerization interface were important for DNA binding and some of them decreased the nuclear–cytoplasm ratio of NFAT5. To understand the mechanisms of these mutations, we model their effect on protein dynamics and DNA binding. We show that the NFAT5 complex without DNA is much more flexible than the complex with DNA. Moreover, DNA binding considerably stabilizes the overall dimeric complex and the NFAT5 dimer is only marginally stable in the absence of DNA. Two sets of NFAT5 mutations from the same DNA-binding loop are found to have different mechanisms of specific and nonspecific binding to DNA. The R217A/E223A/R226A (R293A/E299A/R302A using isoform c numbering) mutant is characterized by significantly compromised binding to DNA and higher complex flexibility. On the contrary, the T222D (T298D in isoform c) mutation, a potential phosphomimetic mutation, makes the overall complex more rigid and does not significantly affect the DNA binding. Therefore, the reduced nuclear–cytoplasm ratio of NFAT5 can be attributed to reduced binding to DNA for the triple mutant, while the T222D mutant suggests an additional mechanism at work.
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