Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50.
To assess the importance of residue 50 in determining the binding specificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains containing glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-type engrailed homeodomain, TAATTA was identified as a high-affinity, consensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The half-life and affinity of the complex between the QK50 protein and a DNA site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein and a TAATTA site. This suggests that Lys50 forms a more favorable interaction with the TAATCC DNA than Gln50 does with the TAATTA site. In fact, the wild-type Gln50 side chain (which forms a hydrophobic interaction with the last A:T base pair of the TAATTA site in the cocrystal structure [Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B., & Pabo, C. O. (1990) Cell 63, 579-590]) appears to play only a small role in determining binding affinity and specificity for the TAATTA site, as the QA50 mutant has only a 2-fold reduced affinity for the TAATTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gln50 must be involved in establishing the differential binding specificity of the engrailed homeodomain.
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
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D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003196:Computer Graphics; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism; D029721:Drosophila Proteins; D005813:Genes, Synthetic; D018398:Homeodomain Proteins; D007301:Insect Hormones / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D007301:Insect Hormones; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA