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J. Biol. Chem., Vol. 283, Issue 48, 33321-33328, November 28, 2008

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The Solution Structure of DNA-free Pax-8 Paired Box Domain Accounts for Redox Regulation of Transcriptional Activity in the Pax Protein Family^*

Luca Codutti, Hugo van Ingen, Carlo Vascotto, Federico Fogolari^¶, Alessandra Corazza^¶, Gianluca Tell, Franco Quadrifoglio, Paolo Viglino^¶, Rolf Boelens, and Gennaro Esposito^¶1

From the Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Udine, p.le Kolbe 4, 33100 Udine, Italy, the Bijvoet Center for Biomolecular Research, Padualaan 8, 3584 CH Utrecht, The Netherlands, and the ^¶INBB (Istituto Nazionale Biostrutture e Biosistemi), Viale Medaglie d'Oro 35, 00100 Roma, Italy

Pax-8 is a transcription factor belonging to the PAX genes superfamily and its crucial role has been proven both in embryo and in the adult organism. Pax-8 activity is regulated via a redoxbased mechanism centered on the glutathionylation of specific cysteines in the N-terminal region (Cys⁴⁵ and Cys⁵⁷). These residues belong to a highly evolutionary conserved DNA binding site: the Paired Box (Prd) domain. Crystallographic protein-DNA complexes of the homologues Pax-6 and Pax-5 showed a bipartite Prd domain consisting of two helix-turn-helix (HTH) motifs separated by an extended linker region. Here, by means of nuclear magnetic resonance, we show for the first time that the HTH motifs are largely defined in the unbound Pax-8 Prd domain. Our findings contrast with previous induced fit models, in which Pax-8 is supposed to largely fold upon DNA binding. Importantly, our data provide the structural basis for the enhanced chemical reactivity of residues Cys⁴⁵ and Cys⁵⁷ and explain clinical missense mutations that are not obviously related to the DNA binding interface of the paired box domain. Finally, sequence conservation suggests that our findings could be a general feature of the Pax family transcription factors.

Received for publication, July 25, 2008 , and in revised form, September 30, 2008.

The atomic coordinates and structure factors (code 2K27) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Italian Ministry for University and Research Grants COFIN 2006058958, FIRB RBNE03PX83, and FIRB RBNE03B8KK, European Union Grant LSHM-CT-2005-037525, and European Union project "EU-NMR-European Network of Research Infrastructures for Providing Access & Technological Advancement in Bio-NMR" FP-2005-RII3 Contract 026145. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6, Tables S1 and S2, additional text, and references.

¹ To whom correspondence should be addressed. Fax: 390432494301; E-mail: gesposito{at}mail.dstb.uniud.it.

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