Redox potential controls the structure and DNA binding activity of the paired domain.

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox 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 therefore different target genes.