Dominant negative dimerization of a mutant homeodomain protein in Axenfeld-Rieger syndrome

Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.     

Solution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome

We have determined the solution structure of a complex containing the K50 class homeodomain Pituitary homeobox protein 2 (PITX2) bound to its consensus DNA site (TAATCC). Previous studies have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains. Although structures of several homeodomain-DNA complexes have been determined, this is the first structure of a native K50 class homeodomain. The only K50 homeodomain structure determined previously is an X-ray crystal structure of an altered specificity mutant, Engrailed Q50K (EnQ50K). Analysis of the NMR structure of the PITX2 homeodomain indicates that the lysine at position 50 makes contacts with two guanines on the antisense strand of the DNA, adjacent to the TAAT core DNA sequence, consistent with the structure of EnQ50K. Our evidence suggests that this side chain may make fluctuating interactions with the DNA, which is complementary to the crystal data for EnQ50K. There are differences in the tertiary structure between the native K50 structure and that of EnQ50K, which may explain differences in affinity and specificity between these proteins. Mutations in the human PITX2 gene are responsible for Rieger syndrome, an autosomal dominant disorder. Analysis of the residues mutated in Rieger syndrome indicates that many of these residues are involved in DNA binding, while others are involved in formation of the hydrophobic core of the protein. Overall, the role of K50 in homeodomain recognition is further clarified, and the results indicate that native K50 homeodomains may exhibit differences from altered specificity mutants.