Crystal structure of the human Pax6 paired domain-DNA complex reveals specific roles for the linker region and carboxy-terminal subdomain in DNA binding

Abstract

Pax6, a transcription factor containing the bipartite paired DNA-binding domain, has critical roles in development of the eye, nose, pancreas, and central nervous system. The 2.5 Å structure of the human Pax6 paired domain with its optimal 26-bp site reveals extensive DNA contacts from the amino-terminal subdomain, the linker region, and the carboxy-terminal subdomain. The Pax6 structure not only confirms the docking arrangement of the amino-terminal subdomain as seen in cocrystals of the Drosophila Prd Pax protein, but also reveals some interesting differences in this region and helps explain the sequence specificity of paired domain-DNA recognition. In addition, this structure gives the first detailed information about how the paired linker region and carboxy-terminal subdomain contact DNA. The extended linker makes minor groove contacts over an 8-bp region, and the carboxy-terminal helix-turn-helix unit makes base contacts in the major groove. The structure and docking arrangement of the carboxy-terminal subdomain of Pax6 is remarkably similar to that of the amino-terminal subdomain, and there is an approximate twofold symmetry axis relating the polypeptide backbones of these two helix-turn-helix units. Our structure of the Pax6 paired domain-DNA complex provides a framework for understanding paired domain-DNA interactions, for analyzing mutations that map in the linker and carboxy-terminal regions of the paired domain, and for modeling protein-protein interactions of the Pax family proteins.

A missense mutation in PAX9 in a family with distinct phenotype of oligodontia

“…In PAX6, homologous Arg26 is localized in the first a-helix of the N-terminal subdomain of the paired domain and is one of the residues that directly contacts DNA. 23 A mutation of this arginine is reported in a family with various anterior segment malformations of the eye. 18 As mutations in the human PAX6 typically produce aniridia, the relatively milder phenotypes of the reported R26G mutation suggest that it creates a hypomorphic allele.…”

“…13,22 For PAX6, the crystallographic structure of the paired domain has been resolved. 23 Sequence comparison shows strong homology, with a 75% amino-acid identity in the paired box between PAX9 and PAX6. In PAX6, homologous Arg26 is localized in the first a-helix of the N-terminal subdomain of the paired domain and is one of the residues that directly contacts DNA.…”

Analysis of the transforming and growth suppressive activities of the PAX3-FKHR oncoprotein

“…Within the paired box, there are two subdomains, an N-terminal subdomain and a C-terminal subdomain, linked by a b-sheet linker (Xu et al, 1999). The glycine at codon 48 is located in the N-subdomain, and has a direct contact with the DNA in its minor groove.…”

Molecular analysis of a human PAX6 homeobox mutant

“…We used two radiolabelled double-stranded oligonucleotide target probes: P3, containing the HD-binding site 29 and Cr, containing the PD-binding site 30 ( Figure 1a). The R242T mutant peptide reveals the same binding activity to the P3 as the wild-type HD, as shown in Figure 1b.…”

“…25 Electophoretic mobility shift assays DNA-binding properties of PAX6 wild-type and mutant peptides were investigated using a series of doublestranded oligonucleotides (top strand only is shown): P3(5 0 -TCGAGGGCATCAGGATGCTAATTGGATTAGCAT CCGATCGGG-3 0 ) and Cr (5 0 -AAGCATTTTCACGCATGAG TGCACAG-3 0 ), which contain consensus DNA-binding sequences for PAX6 HD and PD, respectively. 29,30 The oligonucleotides were radiolabelled at the 5 0 end terminal by incubation with [g-32P]-dATP. Protein and DNA (both at the final concentration of 0.1 mM) were incubated for 30 min at room temperature in a total volume of 20 ml, using a buffer containing 20 mM Tris-HCl (pH 7.6), 75 mM KCl, 0.25 mg/ml bovine serum albumin (BSA), 5 mM dithiotreitol (DTT), 10 mg/ml calf thymus DNA, and 10% glycerol.…”