CONSTRUCTION, PURIFICATION, AND CHARACTERIZATION OF A HYBRID PROTEIN COMPRISING THE DNA-BINDING DOMAIN OF THE LEXA REPRESSOR AND THE JUN LEUCINE ZIPPER - A CIRCULAR-DICHROISM AND MUTAGENESIS STUDY

An increasing number of eukaryotic transcription factors interacting specifically with DNA comprise a dimerization motif called the "leucine zipper". These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif. The leucine zipper of the oncoprotein Jun is particular in that Jun may form homodimers as well as heterodimers with the oncoprotein Fos, which are however more stable than the Jun-Jun homodimers. Leucine zipper dimerization is thought to occur through a colied-coli arrangement of parallel alpha-helices, but the rules governing the specificity of homo- and/or heterodimerization are still largely unknown. To address this question in the case of the Jun leucine zipper, we constructed a fusion protein containing the amino-terminal DNA binding domain of the LexA repressor from Escherichia coli fused to the Jun leucine zipper. This hybrid protein (LexA-JunZip) is stable in E. coli and confers much tighter repression in vivo than the DNA binding domain of LexA alone. DNA binding competition experiments with synthetic Jun and Fos leucine zipper peptides in vitro showed that the leucine zipper mediated dimerization of LexA-JunZip is essential for DNA binding of the fusion protein. The purified LexA-JunZip protein dimerizes in vitro with a dimerization constant of 2 x 10(7) M-1 at 5-degrees-C. Dimerization is very sensitive to temperature, since the dimerization constant drops at 20-degrees-C to 2 x 10(6) M-1 and at 30-degrees-C to only 3 x 10(5) M-1. The heptad repeat (a,b,c,d,e,f,g)n within leucine zipper proteins comprises leucine side chains in position d and generally another hydrophobic side chain in position a. This hydrophobic interface confers probably most of the dimerization free energy, but it has been suggested that the interaction between residues of opposite charge in positions e and g might define the specificity of leucine zipper assembly. To address this question, we have mutated simultaneously and randomly one g and two e codons. It turns out that the Jun leucine zipper prefers in fact hydrophobic side chains in the chosen positions and that the potential charge-charge interactions between positions e and g within the same heptad are not necessarily favorable.