NMR studies of the pbx1 TALE homeodomain protein free in solution and bound to DNA: proposal for a mechanism of HoxB1-pbx1-DNA complex assembly

Abstract

The Hox homeodomain proteins are transcription factors involved in developmental regulation. Many of the vertebrate Hox proteins bind DNA cooperatively with the Pbx1 homeodomain protein. The crystal structure of a human HoxB1-Pbx1-DNA ternary complex revealed that interactions between the two proteins are mediated by the HoxB1 hexapeptide, which inserts into a hydrophobic pocket in Pbx1. It was also found that the Pbx1 DNA-binding domain is larger than the canonical three-helix homeodomain, containing an additional α-helix that is joined to the C terminus of the homeodomain by a turn of 310 helix. These extra C-terminal residues had previously been shown to augment the cooperative interaction of Pbx1 with Hox partners, as well as enhancing the DNA binding of monomeric Pbx1. In order to characterize the role of the fourth Pbx1 helix in greater detail, we have examined the backbone structure of the enlarged Pbx1 DNA-binding domain in solution by 1H, 15N and 13C multidimensional NMR spectroscopy. Our results show that the additional α-helix of Pbx1 is unfolded when the protein is free in solution and that its folding is triggered by binding of Pbx1 to DNA. In contrast, no change in conformation is observed upon mixing the HoxB1 protein with Pbx1 in the absence of DNA. This study suggests a model for the assembly of a stable HoxB1-Pbx1-DNA ternary complex.