Creating temperature-sensitive winged helix transcription factors - Amino acids that stabilize the DNA binding domain of HNF3

Winged helix transcription factors contain two polypeptide loops, or "wings ," that make minor groove contacts with DNA from either side of a three-hel ix bundle that binds the DNA major groove. While wing 1 is stabilized by a beta-sheet, parameters that stabilize wing 2 are unknown. Herein we identif y two bulky aromatic residues in wing 2 that stabilize the loop structure a nd, thereby, the entire protein's DNA binding and transcriptional stimulato ry activity by interacting with other residues in the three-helix bundle. M utations of these wing 2 residues create proteins that are temperature-sens itive for transcriptional activity. Aromatic and/or hydrophobic residues ar e highly conserved among the 150 known winged helix proteins, suggesting co nserved function. We suggest that the winged helix structure evolved by the acquisition of aromatic and/or hydrophobic residues in distal polypeptide sequences that helped stabilize the association of a protein loop (wing 2) with the three helix bundle, thereby enhancing DNA binding.