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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• 作者：Laura Meier-Andrejszki ; Saa Bjeli ; Jean-Franç ; ois Naud ; Pierre Lavigne ; Ilian Jelesarov
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 30, 2007
• 年：2007
• 卷：46
• 期：43
• 页码：12427 - 12440
• 全文大小：340K
• 年卷期：v.46,no.43(October 30, 2007)
• ISSN：1520-4995

文摘

The Myc/Mad/Max network of dimeric basic region-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factors bind to enhancer box sequences (E-box) in the promotors of a large set ofgenes that control cell metabolism, proliferation, and differentiation. Max (Myc-associated factor X) isthe obligate heterodimerization partner of Myc and Mad proteins. On the other hand, Max is the onlymember of the family capable of forming a stable homodimer. As part of the transcriptional regulationmechanism, Myc/Max and Mad/Max heterodimers and Max homodimers are thought to compete for bindingto the E-box target sequences. E-box recognition is structurally supported by the b-HLH-LZ structuralmotif, which also promotes dimerization. However, the actual dimerization and heterodimerization constantsof the complete gene products and their affinities for E-box sequences are not known. Also, the detailedthermodynamic 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 bythe Myc/Mad/Max network. Here, we report the first in-depth thermodynamic characterization of thestability 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 determinedthe dimerization constant of Max p21 in the low micromolar range, and the Max p21/E-box complexdissociation constant in the low nanomolar range at 37 C. The association is driven by a large exothermiceffect, which is partly compensated by entropic factors. The energetic contribution to binding affinity ofseven highly conserved residues that contact the DNA was probed by X-to-Ala mutagenesis. The resultsdemonstrate that high binding affinity critically relies on the side chain of Arg 26. Furthermore, themutational analysis points to the important role of the persistent helical turn that comprises this residueat the junction of the basic region and helix H1. Altogether, the study supports the idea that Max p21 canbind E-box sequences in vivo and likely participates directly in the regulation of transcription as homodimer.