Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

The yeast a2 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 c~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 ~2 is structurally related to prototypical members of the homeo domain family. We used solution ~H and lSN nuclear magnetic resonance (NMR) spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of ~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 ,Y2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3J~N_H. coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the c~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 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 a2 and that of higher eukaryotes, the secondary structures of these proteins are well conserved.