The paired domain of Pax3 contains a putative homeodomain interaction pocket defined by cysteine scanning mutagenesis.
Pax3 is a transcription factor that plays an important regulatory role during neurogenesis, myogenesis, and formation of neural crest cell derived structures. Pax3 has two DNA binding domains, a paired domain (PD) and paired-type homeodomain (HD) that show complete interdependence for DNA binding, with mutations in one domain impairing DNA binding by the other domain. Cooperative interactions between the PD and HD of Pax3 suggest that the two domains may physically interact for DNA binding. Site-specific modification with thiol reagents in single cysteine Pax3 mutants was used to determine which segment of the PD may interact with the HD. Twenty-four single cysteine mutants were independently introduced in the second alpha-helix (alpha2, positions 59-80) and in the beta-hairpin structure (positions 40-41) at the amino terminal portion of the PD. These mutants were tested for their ability to bind to PD (P6CON, P3OPT) and HD-specific DNA targets (P2), and the effect of treatment with N-ethylmaleimide on these binding properties was established. In the PD, single cysteine mutants CL/Q40C, CL/I59C, CL/V60C, CL/P69C, CL/S70C, CL/I72C, CL/S73C, CL/L76C, CL/V78C, and CL/S79C displayed NEM sensitive DNA binding toward both PD and HD targets. Three PD mutants (CL/L41C, CL/A63C, and CL/H64C) showed unusual behavior, with DNA binding to PD targets being NEM insensitive while DNA binding by the HD was abrogated by NEM treatment. Three-dimensional modeling of the NEM sensitive PD cysteine mutants reveal that they are not randomly distributed, but rather that they cluster in a hydrophobic pocket. We propose that this hydrophobic pocket may serve as a docking site for the HD during DNA binding by the intact protein.
Apuzzo
Sergio
S
Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.
Gros
Philippe
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005033:Ethylmaleimide / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D003545:Cysteine; D005033:Ethylmaleimide