Fig. 2. Structural and functional analysis of the HOXA13 DNA-binding domain. (A) The amino acid sequence of the A13-DBD peptide is presented above. (Left panel) Structural analysis of the A13-DBD peptide by circular dichroism spectroscopy indicates that the peptide folds into a stable -helical DNA-binding motif at 4°C (blue curve). At 60°C, the majority of the -helical content is lost by thermal denaturation (red curve). (Right panel) Thermal stability measurements of the A13-DBD peptide showed that the -helical conformation is maintained between 4 and 25°C. At temperatures higher than 25°C, the peptide cooperatively transforms to its denatured conformation. (B-E) A13-DBD peptide exhibits specific binding for the DNA regions present upstream of Bmp2 and Bmp7. Asterisks denote unbound radiolabeled DNA, arrowheads denote A13-DBD-DNA complexes (B,C,E). (B) Quantitation of the A13-DBD affinity for the bound DNA regions using increasing concentrations (black triangles) of cold competitor DNA (0, 150, 300 and 750 nM) revealed differential A13-DBD affinities for each of the bound sites. (C) The Bmp2C2 region requires 2-fold greater concentrations of A13-DBD to affect its electrophoretic mobility. (D) Radiolabeled control DNA sequences exhibited no change in electrophoretic mobility when incubated with 4-fold (2 µM) higher concentrations of the A13-DBD peptide. (E) A13-DBD binding specificity was confirmed using the Bmp7C1 binding site, which could not be displaced from the A13-DBD peptide by using 750 nM concentrations of the unlabeled negative control DNA (cNC), but is completely displaced using 750 nM unlabeled Bmp7C1 competitor DNA (cBMP7C1). (F) Analysis of the DNA sequences bound by the A13-DBD reveals a novel series of HOX binding sites. The core TAAT site is designated in bold type.