Thermodynamics of b-HLH-LZ protein binding to DNA: The energetic importance of protein-DNA contacts in site-specific e-box recognition by the complete gene product of the max p21 transcription factor
Thermodynamics of b-HLH-LZ protein binding to DNA: The energetic importance of protein-DNA contacts in site-specific e-box recognition by the complete gene product of the max p21 transcription factor
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DOI：
10.1021/bi701081q
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发表时间：
2007-10-30
期刊：
影响因子：
2.9
通讯作者：
Jelesarov, Ilian
中科院分区：
文献类型：
--
作者：
Meier-Andrejszki, Laura;Bjelic, Sasa;Jelesarov, Ilian
二聚体碱性区-螺旋-环-螺旋-亮氨酸拉链（bHLH-LZ）转录因子的Myc/Mad/Max网络与控制细胞代谢、增殖和分化的大量基因启动子中的增强子盒序列（E-box）结合。Max （Myc相关因子X）是Myc和Mad蛋白的专性异二聚化伙伴。另一方面，Max是家族中唯一能够形成稳定的同型二聚体的成员。作为转录调控机制的一部分，Myc/Max和Mad/Max异源二聚体和Max同型二聚体被认为会竞争与E-box靶序列的结合。b-HLH-LZ结构基序在结构上支持E-box识别，这也促进了二聚化。然而，完整基因产物的实际二聚化和异二聚化常数及其对E-box序列的亲和力尚不清楚。此外，这些转录因子结合DNA的详细热力学表征尚未完成。这些知识对于完全理解Myc/Mad/Max网络进行的转录调控是必要的。在这里，我们报道了Myc/Mad/Max家族全长基因产物，即Max蛋白异构体p21 （Max p21）的稳定性和特异性DNA结合的首次深入热力学表征。利用量热法（DSC和ITC）测定了Max p21在低微摩尔范围内的二聚化常数，以及Max p21/E-box复合物在37℃时低纳摩尔范围内的解离常数。通过X-to-Ala诱变，探索了7个高度保守残基与DNA的结合亲和力的能量贡献。结果表明，高结合亲和性主要依赖于Arg 26的侧链。此外，突变分析指出了持久的螺旋转向的重要作用，包括在基本区和螺旋H1的连接处的残基。总之，该研究支持了Max p21可以在体内结合E-box序列，并可能作为同型二聚体直接参与转录调控的观点。
The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (bHLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set of genes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) is the obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the only member of the family capable of forming a stable homodimer. As part of the transcriptional regulation mechanism, Myc/Max and Mad/Max heterodimers and Max homodimers are thought to compete for binding to the E-box target sequences. E-box recognition is structurally supported by the b-HLH-LZ structural motif, which also promotes dimerization. However, the actual dimerization and heterodimerization constants of the complete gene products and their affinities for, E-box sequences are not known. Also, the detailed thermodynamic characterization of DNA binding by these transcription factors has not been done yet. Such knowledge is necessary for complete understanding of the transcriptional regulation carried out by the Myc/Mad/Max network. Here, we report the first in-depth thermodynamic characterization of the stability and specific DNA binding of a full length gene product of the Myc/Mad/Max family, namely, Max protein isoform p21 (Max p21). Using calorimetric methods (DSC and ITC) we have determined the dimerization constant of Max p21 in the low micromolar range, and the Max p21/E-box complex dissociation constant in the low nanomolar range at 37 degrees C. The association is driven by a large exothermic effect, which is partly compensated by entropic factors. The energetic contribution to binding affinity of seven highly conserved residues that contact the DNA was probed by X-to-Ala mutagenesis. The results demonstrate that high binding affinity critically relies on the side chain of Arg 26. Furthermore, the mutational analysis points to the important role of the persistent helical turn that comprises this residue at the junction of the basic region and helix H1. Altogether, the study supports the idea that Max p21 can bind E-box sequences in vivo and likely participates directly in the regulation of transcription as homodimer.