The GCN4 bZIP can bind to noncognate gene regulatory sequences

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 microM 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, alpha-helical proteins with desired DNA recognition properties.     

The SKN-1 amino-terminal arm is a DNA specificity segment

The Caenorhabditis elegans SKN-1 protein binds DNA through a basic region like those of bZIP proteins and through a flexible amino-terminal arm segment similar to those with which numerous helix-turn-helix proteins bind to bases in the minor groove. A recent X-ray crystallographic structure suggests that the SKN-1 amino-terminal arm provides only nonspecific DNA binding. In this study, however, we demonstrate that this segment mediates recognition of an AT-rich element that is part of the preferred SKN-1 binding site and thereby significantly increases the sequence specificity with which SKN-1 binds DNA. Mutagenesis experiments show that multiple amino acid residues within the arm are involved in binding. These residues provide binding affinity through distinct but partially redundant interactions and enhance specificity by discriminating against alternate sites. The AT-rich element minor groove is important for binding of the arm, which appears to affect DNA conformation in this region. This conformational effect does not seem to involve DNA bending, however, because the arm does not appear to affect a modest DNA bend that is induced by SKN-1. The data illustrate an example of how a small, flexible protein segment can make an important contribution to DNA binding specificity through multiple interactions and mechanisms.     

Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR

The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.