Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding.
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.
Apuzzo
Sergio
S
Department of Biochemistry and McGill Cancer Center, McGill University, Quebec H1E 1S9, Canada. sapuzz@po-box.mcgill.ca
Abdelhakim
Aliaa
A
Fortin
Anouk S
AS
Gros
Philippe
P
D000595:Amino Acid Sequence; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015951:Factor Xa / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D020239:Receptor Cross-Talk; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C068686:Pax3 protein, mouse; D004247:DNA; D015951:Factor Xa