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J. Biol. Chem., Vol. 283, Issue 7, 4352-4363, February 15, 2008

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Structure-Function Analysis of the THAP Zinc Finger of THAP1, a Large C2CH DNA-binding Module Linked to Rb/E2F Pathways^*

Damien Bessière¹, Chrystelle Lacroix^¶1, Sébastien Campagne, Vincent Ecochard^¶, Valérie Guillet^||, Lionel Mourey^||, Frédéric Lopez^**, Jerzy Czaplicki, Pascal Demange, Alain Milon, Jean-Philippe Girard^¶2, and Virginie Gervais³

From the University of Toulouse, Institute of Pharmacology and Structural Biology, the Laboratory of NMR and Protein-Membrane Interactions, the ^¶Laboratory of Vascular Biology, and the ^||Laboratory of Structural Biophysics, IPBS-CNRS-UPS, 31077 Toulouse, France and ^**INSERM-IFR31, CHU Rangueil, 31059 Toulouse, France

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel β-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.

Received for publication, September 10, 2007 , and in revised form, October 24, 2007.

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

Chemical Shift information for the THAP domains (Met¹-Lys⁹⁰) and (Met¹-Phe⁸¹) are available from the BioMagRes Data Bank (http://www.bmrb.wisc.edu), under the accession numbers 15300 and 15289, respectively.

^* This work was supported in part by the French Research Ministry (ACI BCMS), Ligue Nationale Contre le Cancer (Equipe labelisée, to J. P. G.) and by FRM (to D. B.) and ARC (to C. L.). 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 Fig. S1.

¹ Both authors contributed equally to the work.

² To whom correspondence may be addressed: University of Toulouse, Inst. of Pharmacology and Structural Biology, IPBS-CNRS-UPS, Toulouse, France. E-mail: jean-philippe.girard{at}ipbs.fr.

³ To whom correspondence may be addressed: University of Toulouse, Inst. of Pharmacology and Structural Biology, IPBS-CNRS-UPS, Toulouse, France. E-mail: virginie.gervais{at}ipbs.fr.

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