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J. Biol. Chem., Vol. 278, Issue 41, 40252-40261, October 10, 2003

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Xeroderma Pigmentosum Group C Protein Possesses a High Affinity Binding Site to Human Centrin 2 and Calmodulin^*

Aurel Popescu , Simona Miron , Yves Blouquit, Patricia Duchambon, Petya Christova ¶ and Constantin T. Craescu ||

From the INSERM U350 and Institut Curie-Recherche, Centre Universitaire, Batiments 110-112, 91405 Orsay, France

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca²⁺-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca²⁺ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (K_a = 2.2 x 10⁸ M^-1, stoichiometry 1:1) with HsCen2 in a Ca²⁺-dependent manner. This peptide also binds, with a similar affinity (K_a = 1.1 x 10⁸ M^-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC.

Received for publication, March 12, 2003 , and in revised form, July 29, 2003.

^* This work was supported in part by the Institut Curie, INSERM, and CNRS. 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.

Present address: Faculty of Physics, University of Bucharest, 077125 Bucharest, Romania.

Recipient of the European Marie Curie fellowship.

^¶ Present address: Inst. of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

^|| To whom correspondence should be addressed: INSERM U350 and Institut Curie-Recherche, Centre Universitaire, Batiments 110-112, 91405 Orsay Cedex, France. Tel.: 33-1-69-86-31-63; Fax: 33-1-69-07-53-27; E-mail: gil.craescu{at}curie.u-psud.fr.

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