Abstract
MAp19 is an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB1-epidermal growth factor (EGF) segment of MASP-2, plus four additional residues at its C-terminal end. Like full-length MASP-2, it forms Ca(2+)-dependent complexes with mannan-binding lectin (MBL) and L-ficolin. The x-ray structure of human MAp19 was solved to a resolution of 2.5 A. It shows a head to tail homodimer held together by interactions between the CUB1 module of one monomer and the EGF module of its counterpart. A Ca(2+) ion bound to each EGF module stabilizes the dimer interfaces. A second Ca(2+) ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu(52), Asp(60), Asp(105), Ser(107), Asn(108), and a water molecule. Compared with its counterpart in human C1s, the N-terminal end of the MAp19 CUB1 module contains a 7-residue extension that forms additional inter-monomer contacts. To identify the residues involved in the interaction of MAp19 with MBL and L-ficolin, point mutants were generated and their binding ability was determined using surface plasmon resonance spectroscopy. Six mutations at Tyr(59), Asp(60), Glu(83), Asp(105), Tyr(106), and Glu(109) either strongly decreased or abolished interaction with both MBL and L-ficolin. These mutations map a common binding site for these proteins located at the distal end of each CUB1 module and stabilized by the Ca(2+) ion.
Highlights
Studies performed over the past decade have led to the discovery of a novel route of complement activation termed the lectin pathway, which is increasingly recognized as an important component of innate antimicrobial host defense
The crystal structure of MBL-associated protein 19 (MAp19) was solved by molecular replacement using the rat MASP-2 CUB1-epidermal growth factor (EGF) structure (21) as a search model, and refined to 2.5-Å resolution
The x-ray structure of human MAp19 determined in the present study provides a third example of a CUB-EGF domain structure, allowing a comparison with those determined recently for human C1s (22) and rat MASP-2 (21)
Summary
Proteins—MBL and L-ficolin were purified from human serum as described by Zundel et al (18) and Cseh et al (17), respectively. Recombinant wild-type MAp19 and its variants were expressed using a baculovirus/insect cell system. Whereas initial searches using the human C1s CUB1EGF structure (22) were unsuccessful, a molecular replacement solution was obtained using the CUB1-EGF fragment of rat MASP-2 in the dimeric form (Protein Data Bank accession code 1NT0) (21). Binding of the wild-type and mutant MAp19 species was measured over 16,000 resonance units of immobilized L-ficolin or 9,000 resonance units of immobilized MBL, at a flow rate of 20 l/min in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, containing 0.005% surfactant P20 (BIAcore AB). Each MAp19 variant was analyzed at six different concentrations, ranging from 5 to 60 nM for wild-type MAp19 and all mutants except D60A and E83A (25–250 nM), E109A (50 –500 nM), Y59A, D105G, and Y106A (100 –1200 nM)
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