The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences:  Quantitative Investigation of Binding at Full and Half Sites

Abstract

: 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).

The bZIP Targets Overlapping DNA Subsites within a Half-Site, Resulting in Increased Binding Affinities

“…We initially observed that the wt bZIP displays significant affinity for the C/EBP site only 100-fold weaker than that at the cognate AP-1 and CRE sites [both sites give a K d of 1.6 × 10 −16 M 2 ; previous data recalculated as dimeric dissociation constants (10)]. In contrast, the affinities at other noncognate sites, including the Arnt E-box site, are reduced ≥2000-fold (Table 1) (9).…”

“…In contrast, the affinities at other noncognate sites, including the Arnt E-box site, are reduced ≥2000-fold (Table 1) (9). Furthermore, the affinity of the wt bZIP at the Arnt E-box site increases ∼30-fold from that at its half-site; likewise, the affinity of the wt bZIP at the AP-1 site increases 44-fold from that at its half-site ( K d = 7.0 × 10 −15 M 2 ) (10). These affinity increases are in agreement with measurements by Hollenbeck and Oakley; the affinity of their GCN4 bZIP derivative at the AP-1 and CRE sites increases 30- and 80-fold, respectively, from that at the TGAC half-site (14).…”

“…EMSA was also tried with TKMC buffer and a higher-NaCl buffer [15 mM Tris (pH 7.5), 75 mM NaCl, 1.35 mM KCl, 5 mM MgCl 2 , and 2.5 mM CaCl 2 ]. The wt bZIP has poorer solubility in TKMC and exhibits band broadening in the higher-NaCl buffer (9, 10). Also, we varied the concentration of poly(dI-dC) between 2 and 60 μ g/mL and found no effect on binding activity (9).…”

“…Additionally, the half-site binding affinities at the TTGC and TCAC sequences are the same; however, the wt bZIP targets the C/EBP site (abutting TTGC sequences) with 20-fold stronger affinity than the Arnt E-box site (abutting TCAC sequences) (Figure 1) (10). These observations prompted us to explore further the determinants of affinity for the binding between the wt bZIP and DNA targets.…”

Mutual Cross Talk between the Regulators Hac1 of the Unfolded Protein Response and Gcn4 of the General Amino Acid Control of Saccharomyces cerevisiae

“…Gcn4 can also bind to naturally occurring variants of this sequence (TGATTCA, TGACTCT, TG ACTGA, TGACTAT, and ATGACTCT), and therefore, using a computer algorithm, this consensus site was generalized to RRR WGASTCA (R ϭ purine, W ϭ T or A, and S ϭ G or C) (9). Gcn4 also binds to GCRE half-sites with high affinity in vitro (15,16).…”