Thermal unfolding studies of a leucine zipper domain and its specific DNA complex: implications for scissor's 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 alpha-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 degrees 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 degrees 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 alpha-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 alpha-helical arms.
Weiss
M A
MA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011494:Protein Kinases; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases