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J. Biol. Chem., Vol. 283, Issue 37, 25715-25724, September 12, 2008

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Crystal Structure of the CUB₁-EGF-CUB₂ Domain of Human MASP-1/3 and Identification of Its Interaction Sites with Mannan-binding Lectin and Ficolins^*

Florence Teillet¹, Christine Gaboriaud¹, Monique Lacroix, Lydie Martin, Gérard J. Arlaud, and Nicole M. Thielens²

From the Laboratoire d'Enzymologie Moléculaire and the Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel, CNRS-CEA-UJF, UMR 5075, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France

MASP-1 and MASP-3 are homologous proteases arising from alternative splicing of the MASP1/3 gene. They include an identical CUB₁-EGF-CUB₂-CCP₁-CCP₂ module array prolonged by different serine protease domains at the C-terminal end. The x-ray structure of the CUB₁-EGF-CUB₂ domain of human MASP-1/3, responsible for interaction of MASP-1 and -3 with their partner proteins mannan-binding lectin (MBL) and ficolins, was solved to a resolution of 2.3Å_. The structure shows a head-to-tail homodimer mainly stabilized by hydrophobic interactions between the CUB₁ module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca²⁺ ion bound primarily to both EGF modules stabilizes the intra- and inter-monomer CUB₁-EGF interfaces. Additional Ca²⁺ ions are bound to each CUB₁ and CUB₂ module through six ligands contributed by Glu⁴⁹, Asp⁵⁷, Asp¹⁰², and Ser¹⁰⁴ (CUB₁) and their counterparts Glu²¹⁶, Asp²²⁶, Asp²⁶³, and Ser²⁶⁵ (CUB₂), plus one and two water molecules, respectively. To identify the residues involved in interaction of MASP-1 and -3 with MBL and L- and H-ficolins, 27 point mutants of human MASP-3 were generated, and their binding properties were analyzed using surface plasmon resonance spectroscopy. These mutations map two homologous binding sites contributed by modules CUB₁ and CUB₂, located in close vicinity of their Ca²⁺-binding sites and stabilized by the Ca²⁺ ion. This information allows us to propose a model of the MBL-MASP-1/3 interaction, involving a major electrostatic interaction between two acidic Ca²⁺ ligands of MASP-1/3 and a conserved lysine of MBL. Based on these and other data, a schematic model of a MBL·MASP complex is proposed.

Received for publication, May 9, 2008 , and in revised form, June 11, 2008.

The atomic coordinates and structure factors (code 3DEM) 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 by the Commissariat à l'Energie Atomique, the CNRS, and the Université Joseph Fourier, Grenoble, France. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 33 4 38 78 95 79; Fax: 33 4 38 78 54 94; E-mail: nicole.thielens{at}ibs.fr.

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