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J. Biol. Chem., Vol. 283, Issue 27, 18990-18999, July 4, 2008

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The Structure and Interactions of the Proline-rich Domain of ASPP2^*

Shahar Rotem, Chen Katz, Hadar Benyamini, Mario Lebendiker, Dmitry Veprintsev^¶, Stefan Rüdiger^||1, Tsafi Danieli, and Assaf Friedler²

From the Institute of Chemistry, Protein Expression and Purification Facilities, The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Safra Campus, Givat Ram, Jerusalem 91904, Israel, the ^¶MRC Laboratory of Molecular biology, Hills Road, Cambridge CB2 0QH, United Kingdom, and ^||Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.

Received for publication, October 22, 2007 , and in revised form, April 30, 2008.

^* This work was supported in part by a career development award from the Human Frontier Science Program Organization, by an Excellence Grant from the Israel Cancer Association (to A. F.), and by a Lady Davis fellowship (to H. B.). 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 Tables S1–S3.

¹ Supported by a Marie Curie Excellence Grant of the European Union, a VIDI grant of the Netherlands Science Organization and the High Potential program of Utrecht University.

² To whom correspondence should be addressed: Tel.: 972-2-658-5746; Fax: 972-2-658-5345; E-mail: assaf{at}chem.ch.huji.ac.il.

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