Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.

C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyad-symmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their alpha-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region alpha helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.     

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.     

Single exchanges of amino acids in the basic region change the specificity of N-Myc.

We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity. The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins. Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments. For this we used palindromic target sequences with systematic base pair exchanges. Several mutants with altered DNA binding specificity were identified. Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)). The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are. Exchanges at position -15 broaden the binding specificity. These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence.