Abstract
We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120°. p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters.
Highlights
We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element
We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters
The structural differences between the two 22-bp DNA molecules are obviously due to the different spacers. It remains unclear whether the difference is induced by p63DBD binding because the structures of the unbound DNA molecules are not available
Summary
Protein Production, Crystallization, and Structure Determination—Recombinant p63DBD was produced in Escherichia coli and purified to homogeneity as described previously [10]. The p63DBD crystals in complexes with 22-bp AT, 22-bp GC, and 19-bp DNA molecules diffracted x-rays to 2.5, 2.8, and 4.2 Å resolutions, respectively. The search model for the two structures with 22-bp DNA was a protein dimer of the p63DBD22-bp TA complex including the associated 10-bp DNA [10] (Protein Data Bank accession code 3QYN). A p63DBD dimer in complex with 8-bp DNA derived from half-site of the p63DBD-22-bp GC structure by removing one base pair from each end served as the search model for molecular replacement. Molecular replacement with the p63DBD-19-bp complex as the search model yielded two solutions, related by a 2-fold rotation about the c unit cell axis and a slight translation of ϳ0.5 Å along the b unit cell axis. Temperature factors refinement grouped each p63DBD and DNA double strand. Because of fast dissociation phase rates (koff), the Kd values were determined using a steady state model
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