Structural studies on the PBX homeodomain : effect of binding DNA and HOX cofactors

HOX homeodomain proteins bind short DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity is increased by the formation of cooperative complexes with PBX. PBX is a 430 amino acid protein that contains an atypical homeodomain which is a member of the three amino acid loop extension (TALE) class of homeodomains. The three amino acid loop extension of PBX is required for formation of cooperative complexes with HOX proteins, and contacts a conserved YPWM motif in the HOX partner. The amino acids immediately C-terminal to the PBX homeodomain increase the affinity of the homeodomain for its DNA site and HOX proteins.The structure, protein-DNA and protein-protein interactions of the PBX homeodomain and C-terminal extension were studied using NMR spectroscopy. Solution structures for the PBX homeodomain, a PBX homeodomain-DNA complex, and a ternary PBX-DNA-HOXA1-derived peptide complex were solved. The NMR structures explain the requirement of DNA for the PBX-HOX interaction, and the increased affinity of DNA binding in the presence of the C-terminal extension and HOX YPWM motif.The structure of the free PBX homeodomain demonstrates that an atypical homeodomain has the same fold as a typical homeodomain. Upon binding to DNA, the third alpha-helix of the PBX homeodomain lengthens, forming, with the three amino acid loop extension, a hydrophobic pocket. The HOX YPWM motif binds in this pocket. The HOX peptide itself shows some structure in solution. The interaction between PBX and HOX is therefore an example of "lock and key" binding. The lengthening of the third helix induces the formation of a fourth alpha-helix in the C-terminal extension. This stabilizes the structure of the homeodomain, accounting for the increase in affinity for DNA and the HOX YPWM motif. Both the PBX homeodomain and the extended PBX homeodomain make identical contacts with a 5'-TGAT-3 ' DNA site and a YPWM peptide.Variations in DNA sequence selectivity of heterodimeric PBX-HOX complexes depend on the HOX partner. However, a comparison of five different HOX-derived YPWM peptides showed that each bound to PBX in the same way, differing only in the strength of the association