Homeodomain proteins: what governs their ability to recognize specific DNA sequences?

Deformed (Dfd) and Ultrabithorax (Ubx) are homeodomain proteins from Drosophila melanogaster that exert regulatory effects on gene expression by binding to specific target sites in the fly genome using a helix-turn-helix (HTH) motif. The recognition helices of these two proteins are almost identical and the DNA sequences they recognize are similar, containing a conserved TAAT core sequence flanked by a somewhat variable sequence. Yet the in vivo functions of the two proteins are quite different. We have used the homeodomains of these two proteins and in vitro selected DNA binding sites to characterize the structural details of homeodomain binding to DNA and to understand the basis for the differences in sequence specificity between homeodomains with similar recognition helices. We have employed hydroxyl radical cleavage of DNA to study the positioning of the proteins on the binding sites and have analyzed the effects of missing nucleosides and purine methylation on homeodomain binding. Our results indicate that the positioning of the Ubx and Dfd homeodomains on their binding sites is consistent with reported structures of other homeodomain/DNA complexes. Dfd and Ubx bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. However, we observe striking differences between the two homeodomains in their specific interactions with DNA. Missing nucleosides within the selected binding sites have differential effects on protein binding, which are dependent on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove.