: We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5′-TGACTCA-3′) and cAMP-response element (CRE) (5′-TGACGTCA-3′) but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5′-TTGCGCAA), XRE1 (xenobiotic response element, 5′-TTGCGTGA), HRE (HIF response element, 5′-GCACGTAG), and E-box (5′-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full-and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with K d values of 13 and 12 nM, respectively, to noncognate sites with K d values of 120 nM to low µM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (K d ) 84 nM) to noncognate half sites (all K d values > 2 µM). CD shows that comparable levels of R-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, R-helical proteins with desired DNA recognition properties.In order to examine the relationship between protein structure and DNA-binding function, we exploit the protein R-helix, a structure used ubiquitously for sequence-selective DNA recognition and one that chemists have successfully manipulated in design and synthesis studies for many years (examples include refs 1-8). GCN4 is a dimeric transcriptional regulator that governs histidine biosynthesis in yeast under conditions of amino acid starvation (9). Crystal structures of the basic region/leucine zipper (bZIP) 1 domain of GCN4 bound to the AP-1 site (5′-TGACTCA) (10) and cAMP-response element (CRE) site (5′-TGACGTCA) (11, 12) and the Jun-Fos bZIP heterodimer bound to show that a continuous R-helix provides the basic region interface for binding to specific DNA sites, as well as the leucine zipper coiled coil dimerization structure. The fulllength GCN4 monomer is 281 amino acids, and the bZIP comprises a dimer of ∼60 residue monomers. We previously generated a series of minimalist bZIP proteins comprising alanine-rich variants of the GCN4 basic region fused to the C/EBP leucine zipper: The wt bZIP comprises the native GCN4 basic region. McKnight and co-workers showed that exchanging basic regions and leucine zippers between GCN4 and C/EBP resulted in functional proteins that targeted cognate DNA sites (14), and likewise, we found our wt bZIP hybrid to be functionally equivalent to the GCN4 bZIP (15,16).

We show that a minimalist basic region/leucine zipper (bZIP) hybrid, comprising the yeast GCN4 basic region and C/EBP leucine zipper, can target mammalian and other gene regulatory sequences naturally targeted by other bZIP and basic/helix-loop-helix (bHLH) proteins. We previously reported that this hybrid, wt bZIP, is capable of sequence-specific, high-affinity binding of DNA comparable to that of native GCN4 to the cognate AP-1 and CRE DNA sites. In this work, we used DNase I footprinting and electrophoretic mobility shift assay to show that wt bZIP can also specifically target noncognate gene regulatory sequences: C/EBP (CCAAT/enhancer binding protein, 5′-TTGCGCAA), XRE1 (Xenobiotic response element, 5′-TTGCGTGA), HRE (HIF response element, 5′-GCACGTAG), and the E-box (Enhancer box, 5′-CACGTG). Although wt bZIP still targets AP-1 with strongest affinity, both DNA-binding specificity and affinity are maintained with wt bZIP binding to noncognate gene regulatory sequences: the dissociation constant for wt bZIP in complex with AP-1 is 13 nM, while that for C/EBP is 120 nM, XRE1 240 nM, and E-box and HRE are in the μM range. These results demonstrate that the bZIP possesses the versatility to bind various sequences with varying affinities, illustrating the potential to fine-tune a designed protein's affinity for its DNA target. Thus, the bZIP scaffold may be a powerful tool in design of small, α-helical proteins with desired DNA recognition properties.

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;...

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