Differential DNA-Binding Specificity of the Engrailed Homeodomain: The Role of Residue 50

Sarah E. Ades, Robert T. Sauer

Research output: Contribution to journalArticle

105 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)9187-9194
Number of pages8
JournalBiochemistry
Volume33
Issue number31
DOIs
StatePublished - Aug 1 1994

All Science Journal Classification (ASJC) codes

  • Biochemistry

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