title: The DNA binding specificity of engrailed homeodomain.
abstract: The engrailed gene of Drosophila melanogaster is an integral member of the highly complex cascade which results in a fully developed fruitfly. The gene product of engrailed contains a homeodomain which is responsible for DNA binding via a helix-turn-helix motif. The crystal structure of this 60 amino acid residue domain complexed to DNA is analogous to structures of other homeodomain-DNA complexes, consistent with the high degree of sequence conservation within both protein and DNA. Despite the high degree of homology, homeodomains do exhibit distinct preferences for certain DNA sequences. Such specificity may be at least partly responsible for the interactions necessary for normal development. Using the hydroxyl radical as a chemical probe, we have examined complexes of Engrailed homeodomain with several DNA sequences to determine the protein's binding specificity in solution. We find that Engrailed forms a single, specific complex with a unique DNA binding site which is analogous to the complex seen in the co-crystal structure. In contrast, our chemical probe experiments show that the binding site of Engrailed that was determined by in vitro selection and that also was present in the co-crystal structure contains two possible binding sites. Modification of the sequence of this site to yield single binding sites removes the ambiguity, and results in two different, well-behaved Engrailed-DNA complexes. Our results underscore the utility of chemical probe experiments for defining the variety of modes of interaction of proteins with DNA that can occur in solution, but that might not be apparent in a crystal structure.
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: A
authors.initials: A
authors.lastname: Draganescu
authors.forename: T D
authors.initials: TD
authors.lastname: Tullius
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; C106185:engrailed homeobox proteins; D004247:DNA