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 nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, 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.

Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K d 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60–70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

In our work with designed minimalist proteins based on the bZIP motif, we have found our Histagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

We previously reported that the wt bZIP, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes GCN4 cognate site AP-1 (TGACTCA) but also selectively targets noncognate DNA sites, in particular the C/EBP site (TTGCGCAA). In this work, we used electrophoretic mobility shift assay and DNase I footprinting to investigate the factors driving the high affinity between the wt bZIP and the C/EBP site. We found that on each strand of the C/EBP site, the wt bZIP recognizes two 4 bp subsites, TTGC and TGCG, which overlap to form the effective 5 bp half-site (TTGCG). The affinity of the wt bZIP for the overall 5 bp half-site is ≥10-fold stronger than that for either 4 bp subsite. Our results suggest that interactions of the wt bZIP with both subsites contribute to the strong affinity at the overall 5 bp half-site and, consequently, the C/EBP site. Accordingly, we propose that the wt bZIP undergoes conformational changes to slide between the two overlapping subsites on the same DNA strand and establish sequence-selective contacts with the different subsites. The proposed binding mechanism expands our understanding of what constitutes an actual DNA target site in protein-DNA interactions.The basic region/leucine zipper (bZIP) 1 is the simplest DNA-binding motif used by transcription factors. Complexes of the GCN4 bZIP with the cognate AP-1 and CRE sites show how this motif engages sequence-specific DNA binding (1-5). The bZIP targets DNA as a dimer of short, seamless α-helices: each monomer comprises a basic region for targeting the DNA major groove and a leucine zipper for dimerization via coiled-coil structure. Thus, the bZIP provides a straightforward, native motif for examination of the relationship between protein structure and DNA-binding function.We previously generated the wt bZIP (wild type), a hybrid of the GCN4 basic region and C/ EBP leucine zipper that maintains α-helical structure and DNA-binding function comparable † We express gratitude for funding from the National Institutes of Health (RO1GM069041), the Canadian Foundation for Innovation/ Ontario Innovation Trust (CFI/OIT), the Premier's Research Excellence Award (PREA), and the University of Toronto. SUPPORTING INFORMATION AVAILABLE Target site analyses. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: bZIP, basic region/leucine zipper; CRE, cAMP-response element; C/EBP, CCAAT/enhancer binding protein; Arnt, aryl hydrocarbon receptor nuclear translocator; E-box, enhancer box; bHLH/PAS, basic/helix-loop-helix/Per-Arnt-Sim; EMSA, electrophoretic mobility shift assay; ESI-MS, electrospray ionization mass spectrometry; e-wt bZIP, bacterially expressed wt bZIP; s-wt bZIP, chemically synthesized wt bZIP; HPLC, high-performance liquid chromatography; BSA, bovine serum albumin; DTT, dithiothreitol; T-leap, temperature leap; PAGE, polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; TBE, Trisborate-EDTA; K d , apparent dimeric equilibrium dissociation constant;...

We describe a bacterial reporter system, FRep, for rapid and facile detection of protein-DNA recognition. The bioprobe reporter comprises genes of two fluorescent proteins (FPs) separated by a potential DNA target. If a coexpressed transcription factor binds the DNA target, transcription of the second FP is impeded, resulting in loss of FRET partner. Using ratiometric FRET, we show that evaluation of protein-DNA recognition can be reliably made on bZIP and bHLHZ transcription factors and their DNA targets. FRep displays similar thresholds of detection regarding protein-DNA binding affinities, as compared to well-established electrophoretic and yeast assays, although we observed variations in the intensity of fluorescence signals and detection thresholds that may depend on differences between DNA-binding protein production levels and/or stability in the cell, or the expressed bioprobe linker between the two FPs. FRep can potentially be applied to high-throughput searches of both protein and DNA libraries; in a mock library screen, binding and nonbinding complexes can even be distinguished by visual inspection of colonies on plates. FRep presents notable advantages over existing technologies when applied to assessing protein-DNA interactions in vivo, and this approach has the potential for applications in assaying protein-protein interactions and screening molecules that influence specific macromolecular interactions.