NMR studies of the Pbx1 TALE homeodomain protein free in solution and bound to DNA: Proposal for a mechanism of HoxB1-Pbx1-DMA complex assembly

The Hox homeodomain proteins are transcription factors involved in developm ental regulation. Many of the vertebrate Hox proteins bind DNA cooperativel y with the Pbx1 homeodomain protein. The crystal structure of a human HoxB1 -Pbx1-DNA ternary complex revealed that interactions between the two protei ns 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 large r than the canonical three-helix homeodomain, containing an additional a-he lix that is joined to the C terminus of the homeodomain by a turn of 3(10) heir. 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 th e fourth Pbx1 helix in greater detail, we have examined the backbone struct ure of the enlarged Pbx1 DNA-binding domain in solution by H-1, N-15 and C- 13 multidimensional NMR spectroscopy. Our results show that the additional a-helix of Pbx1 is unfolded when the protein is free in solution and that i ts folding is triggered by binding of Pbx1 to DNA, In contrast, no change i n conformation is observed upon mixing the HoxB1 protein with Pbx1 in the a bsence of DNA. This study suggests a model for the assembly of a stable Hox B1-Pbx1-DNA ternary complex. (C) 1999 Academic Press.