Max-E47, a Designed Minimalist Protein That Targets the E-Box DNA Site in Vivo and in Vitro

• Published in: Journal of the American Chemical Society 2009-06, Vol.131 (22), p.7839-7848
• Main Authors: Xu, Jing, Chen, Gang, De Jong, Antonia T, Shahravan, S. Hesam, Shin, Jumi A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - chemistry; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - genetics; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - metabolism; Dimerization; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; E-Box Elements; Fluorescence Polarization; Helix-Loop-Helix Motifs; Models, Molecular; Molecular Sequence Data; Plasmids - genetics; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Repressor Proteins - chemistry; Repressor Proteins - genetics; Repressor Proteins - metabolism; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptional Activation
• ISSN: 0002-7863; 1520-5126
• 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.