SECONDARY STRUCTURE OF THE HOMEO DOMAIN OF YEAST ALPHA-2 REPRESSOR DETERMINED BY NMR-SPECTROSCOPY

The yeast alpha-2 protein is a regulator of cell type in Saccharomyces cerevisiae. It represses transcription of a set of target genes by binding to an operator located upstream of each of these genes. The alpha-2 protein shares weak sequence similarity with members of the homeo domain family; the homeo domain is a 60-amino-acid segment found in many eukaryotic transcriptional regulators. In this paper we address the question of whether alpha-2 is structurally related to prototypical members of the homeo domain family. We used solution H-1 and N-15 nuclear magnetic resonance (NMR) spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of alpha-2 that contains the homeo domain homology. We have obtained resonance assignments for the backbone protons and nitrogens of the entire 60-residue region of the putative homeo domain and for most of the remainder of the alpha-2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3J(HN-H-alpha) coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the alpha-2 fragment consisting of residues 11-23, 32-42, and 46-60 (corresponding to residues 138-150, 159-169, and 173-187 of the intact protein). The positions of these three helices correspond extremely well to those of the Drosophila Antennapedia (Antp) and engrailed (en) homeo domains, whose three-dimensional structures have recently been determined by NMR spectroscopy and X-ray crystallography, respectively. This study reveals that in spite of low sequence similarity between the homeo domain of alpha-2 and that of higher eukaryotes, the secondary structures of these proteins are well conserved.