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-helix 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 identify two bulky aromatic residues in wing 2 that stabilize the loop structure and, thereby, the entire protein's DNA binding and transcriptional stimulatory activity by interacting with other residues in the three-helix bundle. Mutations of these wing 2 residues create proteins that are temperature-sensitive for transcriptional activity. Aromatic and/or hydrophobic residues are highly conserved among the 150 known winged helix proteins, suggesting conserved 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.
Stevens
K
K
Cell and Developmental Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Cirillo
L
L
Zaret
K S
KS
D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D018257:Helix-Loop-Helix Motifs; D051541:Hepatocyte Nuclear Factor 3-alpha; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D014157:Transcription Factors