<br>Max-E47，一种设计极简的蛋白质，针对 Vivoandin Vitro 中的 E-Box DNA 位点,Journal of the American Chemical Society

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Max-E47，一种设计极简的蛋白质，针对 Vivoandin Vitro 中的 E-Box DNA 位点
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2009-06-10 , DOI: 10.1021/ja901306q
Jing Xu ₁ , Gang Chen , Antonia T De Jong , S Hesam Shahravan , Jumi A Shin

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Max-E47 是一种设计的杂合蛋白，包含 Max DNA 结合基本区和 E47 HLH 二聚化子结构域。在酵母单杂交系统 (Y1H) 中，Max-E47 显示出来自 E-box 位点 5'-CACGTG 的强转录激活作用，该位点是 Myc/Max/Mad 转录因子网络的目标；两个突变体 Max-E47Y 和 Max-E47YF 从 Y1H 中的 E 盒激活更弱。定量荧光各向异性滴定以获得蛋白质的自由能：DNA 结合为天然 MaxbHLHZ、Max-E47 以及与 E-box 位点结合的 Y 和 YF 突变体（14、15、9、和 6 nM），未检测到与非特异性对照双链体的结合。因为这些极简主义的 E-box 结合杂交体没有激活结构域，并且与 c-MycbHLHZ 没有相互作用，如酵母双杂交测定所示，因此它们有可能作为显性失活抑制剂，抑制 E-box 的激活。转录因子（包括 c-Myc/Max 复合体）靶向的响应基因。作为原理验证，我们使用了经过修改的 Y1H，它允许两种争夺 DNA 靶标的蛋白质之间直接竞争，以表明 Max-E47 有效地击败了天然 MaxbHLHZ 的 E-box；从两个突变体观察到较弱的竞争，与 Y1H 结果一致。这些杂交体为进一步探索蛋白质结构和 DNA 结合功能之间的关系提供了一个极简的支架，并可能作为能够靶向 E-box 位点的蛋白质治疗剂或生化探针来应用。

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Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Sitein Vivoandin Vitro

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nanomolar K(d) values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14, 15, 9, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.

更新日期：2009-06-10

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