Xu Jing, Chen Gang, De Jong Antonia T, Shahravan S Hesam, Shin Jumi A
Department of Chemistry, University of Toronto, Mississauga, Ontario L5L 1C6, Canada.
J Am Chem Soc. 2009 Jun 10;131(22):7839-48. doi: 10.1021/ja901306q.
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.
Max-E47是一种设计的杂交蛋白，由Max DNA结合碱性区域和E47 HLH二聚化亚结构域组成。在酵母单杂交系统（Y1H）中，Max-E47在转录因子Myc/Max/Mad网络靶向的E-box位点（5'-CACGTG）处显示出强烈的转录激活；两个突变体Max-E47Y和Max-E47YF在Y1H中从E-box的激活作用较弱。通过定量荧光各向异性滴定以获得蛋白质与DNA结合的自由能，结果显示天然的MaxbHLHZ、Max-E47以及Y和YF突变体与E-box位点结合的K(d)值为低纳摩尔（分别为14、15、9和6 nM），且未检测到与非特异性对照双链体的结合。由于这些极简的、结合E-box的杂交体没有激活结构域，且如酵母双杂交试验所示，它们与c-MycbHLHZ没有相互作用，因此它们有可能作为显性负性抑制剂，抑制包括c-Myc/Max复合物在内的转录因子靶向的E-box反应基因的激活。作为原理验证，我们使用了改良的Y1H，该系统允许两种竞争DNA靶点的蛋白质之间直接竞争，结果表明Max-E47能有效地与天然的MaxbHLHZ竞争E-box；两个突变体的竞争较弱，这与Y1H结果一致。这些杂交体为进一步探索蛋白质结构与DNA结合功能之间的关系提供了一个极简的支架，并且可能作为能够靶向E-box位点的蛋白质治疗剂或生化探针具有应用价值。
