title: Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm.
abstract: The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.
authors.affiliation: Department of Chemistry, University of Michigan-Flint, Flint, MI 48502, USA. rfrazee@umflint.edu
authors.forename: Richard W
authors.initials: RW
authors.lastname: Frazee
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Taylor
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Tullius
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors
chemical_list: D004268:DNA-Binding Proteins; C066676:Dfd protein, Drosophila; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C066177:Ubx protein, Drosophila; D017665:Hydroxyl Radical; D004247:DNA