HETERODIMERIZATION OF THE YEAST HOMEODOMAIN TRANSCRIPTIONAL REGULATORS ALPHA-2 AND A1 INDUCES AN INTERFACIAL HELIX IN ALPHA-2

The homeodomain proteins a1 and alpha 2 act cooperatively to regulate cell type specific genes in yeast. The basis of the cooperativity is a weak interaction between the two proteins which forms heterodimers th at bind DNA tightly and specifically. In this paper, we examine the me chanism of heterodimerization. We show that two relatively small fragm ents of a1 and alpha 2 are capable of heterodimerization and tight DNA binding. The alpha 2 fragment contains the homeodomain followed by th e natural 22 C-terminal amino acids of the protein; these 22 amino aci ds are unstructured in the alpha 2 fragment. The a1 fragment contains only the homeodomain, indicating that the a1 homeodomain mediates both DNA binding and protein-protein interactions with alpha 2. We used is otope-edited NMR spectroscopy to study the interaction in solution of these two fragments. Samples in which only the alpha 2 fragment was un iformly labeled with N-15 allowed us to visualize changes in the NMR s pectra of the alpha 2 fragment produced by heterodimerization. We foun d that the a1 homeodomain perturbs the resonances of only the C-termin al tail of alpha 2; moreover, contact with a1 converts a portion of th is tail (residues 193-203) from its unstructured state to an alpha-hel ix, as determined by J coupling and NOE measurements. Thus the heterod imerization of two homeodomain proteins involves the specific interact ion between a tail of one protein and the homeodomain of the other. Th is interaction is accompanied by the acquisition of secondary structur e in the tail.