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J. Biol. Chem., Vol. 282, Issue 7, 5015-5025, February 16, 2007

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The C2A-C2B Linker Defines the High Affinity Ca²⁺ Binding Mode of Rabphilin-3A^*

Pierre Montaville¹, Christine Schlicker¹, Andrei Leonov, Markus Zweckstetter², George M. Sheldrick³, and Stefan Becker⁴

From the Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, and the Department of Structural Chemistry, University of Göttingen, 37077 Göttingen, Germany

The Ca²⁺ binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca²⁺ binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca²⁺-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca²⁺ ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca²⁺ binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca²⁺-bound state of the C2B domain. In addition, this Ca²⁺ binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca²⁺ binding mode not only shows how a C2 domain increases its intrinsic Ca²⁺ affinity, but also provides the structural base for an atypical protein-Ca²⁺-phospholipid binding mode of rabphilin-3A.

Received for publication, July 14, 2006 , and in revised form, November 29, 2006.

The atomic coordinates and structure factors (code 2CM5 and 2CM6) 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 in part by the Max Planck Society. 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. 1-11 and Tables 1-4.

¹ Both authors contributed equally to this work.

² Supported by Emmy Noether Grant ZW 71/1-5 from the Deutsche Forschungsgemeinschaft.

³ Supported by the Fonds der Chemischen Industrie.

⁴ To whom correspondence should be addressed: Dept. of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Faberg 11, 37077 Göttingen, Germany. Tel.: 49-551-201-2222; Fax: 49-551-201-2202; E-mail: sabe{at}nmr.mpibpc.mpg.de.

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				N. Coudevylle, P. Montaville, A. Leonov, M. Zweckstetter, and S. Becker Structural Determinants for Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Binding by the C2A Domain of Rabphilin-3A J. Biol. Chem., December 19, 2008; 283(51): 35918 - 35928. [Abstract] [Full Text] [PDF]