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

Abstract

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.