Redox Potential Controls the Structure and DNA Binding Activity of the Paired Domain

Gianluca Tell,Andrea Scaloni,Lucia Pellizzari,Silvestro Formisano,Carlo Pucillo,Giuseppe Damante

doi:10.1074/jbc.273.39.25062

Abstract

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.

Highlights

The paired box containing (Pax) genes encode for morphogenic transcription factors and constitute a multigene family conserved from nematodes to vertebrates [1,2,3,4]
The Prd domain of Pax-8 was purified to homogeneity as previously reported [23] with minor modifications, and its structure was confirmed by mass spectrometry
In order to identify whether Pax-8 and Pax-5 polypeptides are sensitive to redox potential in the interaction with their respective DNA-binding sites, the recombinant proteins were treated with fixed amounts of the reducing agent dithiothreitol (DTT) with the oxidizing agent diazene dicarboxylic acid bis[N,N-dimethylamide] or were exposed to prolonged air conditions corresponding to an oxidative environment

Summary

Introduction

The paired box containing (Pax) genes encode for morphogenic transcription factors and constitute a multigene family conserved from nematodes to vertebrates [1,2,3,4]. The resolution of the crystal structure of the Prd domain of the Drosophila paired protein bound to a target DNA sequence confirmed the presence of two structurally distinct and independent subdomains (named PAI and RED, see Ref. 22), both containing a helix-turn-helix motif joined by a linker region [23]. The biological effects of Pax genes are linearly related to the amounts of the functional protein products Both in humans and in mice, abnormalities due to PAX-6 mutations are semidominant; whereas the eye phenotype is more severe in the homozygous state, several abnormalities are evident in the heterozygous state [26]. In addition we observed that nuclear extracts of several cell lines contain a temperature-sensitive activity able to mimic the effects of reducing agents

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Conclusion