DIFFERENTIAL DNA-BINDING SPECIFICITY OF THE ENGRAILED HOMEODOMAIN - THE ROLE OF RESIDUE-50

To assess the importance of residue 50 in determining the binding spec ificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains contai ning glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-typ e engrailed homeodomain, TAATTA was identified as a high-affinity, con sensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The h alf-life and affinity of the complex between the QK50 protein and a DN A site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein an d a TAATTA site. This suggests that Lys50 forms a more favorable inter action with the TAATCC DNA than Gln50 does with the TAATTA site. In fa ct, the wild-type Gln50 side chain (which forms a hydrophobic interact ion with the last A:T base pair of the TAATTA site in the cocrystal st ructure [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 s ite, as the QA50 mutant has only a 2-fold reduced affinity for the TAA TTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gl n50 must be involved in establishing the differential binding specific ity of the engrailed homeodomain.