REDOX POTENTIAL CONTROLS THE STRUCTURE AND DNA-BINDING ACTIVITY OF THE PAIRED DOMAIN

Pax proteins are transcriptional regulators controlling a variety of c ell fates during animal development. This role depends on the intact f unction of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-h elix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-transla tional and post-translational mechanisms. By using Pax-5 and Pax-8 rec ombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cyst eine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reve rsible formation of an intramolecular disulfide bridge involving the c ysteines present in the PAI subdomain, whereas the RED subdomain appea rs quite insensitive to redox potential. Circular dichroism experiment s indicate that only the reduced form of the Prd domain is able to und ergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an act ivity that is able to reduce the Paired domain and, therefore, to cont rol the DNA binding activity of this protein. Immunodepletion of nucle ar extracts demonstrate that the protein Ref-1 contributes to the redo x regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in ''switching'' among different DNA sequences and therefo re different target genes.