Abstract
The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.
Highlights
Pax3 is a member of a family of 9 transcription factors [1], defined by a DNA binding module, the Paired domain (PD),1 that was first identified in the Drosophila protein Paired (Prd) [2]
Construction of Pax3 Mutants Bearing Factor Xa Protease Cleavage Sites—A large body of biochemical and genetic data indicate that the Paired domain (PD) and the Homeo domain (HD) of Pax3 are functionally interdependent with strong cooperativity between the two sites
Possible conformational changes were assessed by protease sensitivity by monitoring accessibility of cleavage sites strategically inserted in the PD and HD of Pax3
Summary
Pax is a member of a family of 9 transcription factors [1], defined by a DNA binding module, the Paired domain (PD), that was first identified in the Drosophila protein Paired (Prd) [2]. Studies in chimeric PAX3 proteins have shown that the PD can modulate DNA binding specificity and dimerization potential of heterologous HDs [37]. Partial proteolytic digestion with enzymes such as trypsin, chymotrypsin, and papain has been used in such studies, delineating the cleavage sites is complicated by the necessity to identify proteolytic fragments by epitope mapping with specific antibodies and/or peptide sequencing [53] Another implementation of this method involves creating recombinant proteins bearing single heterologous protease cleavage sites (such as Factor Xa) inserted at pre-determined positions in individual mutants. Proteolytic products can be identified using antibodies against antigenic epitope engineered at convenient positions In this approach, conformational changes can be studied in a set of recombinant proteins for which structural and functional integrity has been ascertained
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