THERMAL UNFOLDING STUDIES OF A LEUCINE ZIPPER DOMAIN AND ITS SPECIFIC DNA COMPLEX - IMPLICATIONS FOR SCISSORS GRIP RECOGNITION

A newly recognized class of eukaryotic transcription factors is characterized by a bipartite sequence motif, consisting of a C-terminal dimerization region (the leucine zipper) and an N-terminal basic region (which mediates DNA binding). In studies of isolated leucine zipper peptides, the dimerization region has been characterized as a coiled coil of parallel α-helices. To extend these studies to a functional DNA-binding domain, we describe CD studies of the thermal unfolding and refolding of a 58-residue fragment of GCN4, the yeast homologue of the c-Jun protooncoprotein. This fragment, which contains the complete leucine zipper and basic region, retains the DNA-binding properties of the intact protein. The GCN4 DNA-binding domain exhibits two independent helix-coil unfolding transitions. The major transition (midpoint 65 °C) is due to dissociation of the dimer in accord with previous studies of an isolated leucine zipper. A novel pretransition in the temperature range 0–40 °C is also observed, which reflects partial stabilization of the nascent helix in the basic region. Remarkably, complete folding of the basic region as an α-helix requires specific DNA binding, and the protein-DNA complex exhibits a single cooperative unfolding transition. These results support a major feature of the recently proposed “scissor’s grip” model of DNA recognition, in which the basic regions extend from the leucine zipper as bifurcating α-helical arms. © 1990, American Chemical Society. All rights reserved.