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J. Biol. Chem., Vol. 280, Issue 32, 29107-29116, August 12, 2005

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STD and TRNOESY NMR Studies on the Conformation of the Oncogenic Protein -Catenin Containing the Phosphorylated Motif DpSGXXpS Bound to the -TrCP Protein^*

Simon Megy, Gildas Bertho, Josyane Gharbi-Benarous, Nathalie Evrard-Todeschi, Gael Coadou, Emmanuel Ségéral, Catherine Iehle¶, Eric Quéméneur¶, Richard Benarous, and Jean-Pierre Girault||

From the Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), Université René Descartes-Paris V, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France, the Département des Maladies Infectieuses, Institut Cochin, U567-INSERM, UMR 8104 CNRS, Hôpital Cochin-Paris, Bat. G. Roussy, 27 rue du Faubourg St-Jacques, 75014 Paris, France, and the ^¶Commissariat à l'Energie Atomique Valrho, Direction des Sciences du Vivant/Département d'Ingénierie et d'Etude des Protéines/Service de recherche de Biochimie post-génomique et Toxicologie Nucléaire, BP17171, 30207 Bagnols-sur-Cèze, France

-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that -TrCP targets the oncogenic protein -catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from -catenin containing the phosphorylated motif DpSGXXpS (P--Cat^17–48) and -TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the -TrCP binding pocket. The amide and aromatic protons of the His²⁴ and Trp²⁵ residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter -catenin fragment with the -TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated -catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.

Received for publication, February 11, 2005 , and in revised form, May 31, 2005.

^* This work was supported by grants from the Association pour la Recherche sur le Cancer (to E. S.) and the Fondation Recherche Médicale (to S. M.). 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.

^|| To whom correspondence should be addressed. Tel.: 33-1-42-86-21-80; Fax: 33-1-42-86-83-87; E-mail: jean-pierre.girault{at}univ-paris5.fr.

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