Trivalent Recognition Unit of Innate Immunity System: CRYSTAL STRUCTURE OF TRIMERIC HUMAN M-FICOLIN FIBRINOGEN-LIKE DOMAIN

Michikazu Tanio,Shin Kondo,Shigetoshi Sugio,Toshiyuki Kohno

doi:10.1074/jbc.m608627200

Michikazu Tanio, Shin Kondo + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m608627200

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Abstract

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution. Although the FD1 monomer shares a common fold with the fibrinogen gamma fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins.

Highlights

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems
Collectins, such as mannose-binding lectin (MBL), lung surfactant protein A, and surfactant protein D, consist of an N-terminal collagen-like domain and a C-terminal carbohydrate-recognition domain (CRD) that binds to certain carbohydrates such as mannose and GlcNAc Ca2ϩ dependently
The crystal structures of the human fibrinogen ␥ fragment [33] and of the fibrinogen-like domain of Tachypleus tridentatus tachylectin-5A (TL5A) [34] have shown that the P domain contributes to ligand binding

Summary

EXPERIMENTAL PROCEDURES

Purification and Crystallization of FD1—FD1, comprising residues 115–326 of M-ficolin, with the C-terminal peptide containing a c-myc epitope and a His tag, was overexpressed in the yeast Pichia pastoris, purified to homogeneity, and crystallized as described previously [36]. Crystals were obtained at 20 °C by mixing 0.5 ␮l of the protein solution (8 mg mlϪ1 FD1, 8 mM Tris-HCl, pH 8.0, 80 mM NaCl, 4 mM CaCl2 and 20 mM GlcNAc) with 0.5 ␮l of the reservoir solution (100 mM MES, pH 5.6, 320 mM Li2SO4, and 17% (w/v) polyethylene glycol 4000) [36]. The current atomic model gave Rwork and Rfree factors 20.9 and 24.0%, respectively, against all reflections in the resolution range of 30 –1.9 Å (see Table 1). Measurement of Sugar Binding Activity—The purified FD1 was diluted to 0.1 mg mlϪ1 in 200 ␮l of Tris buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5 mM CaCl2) with or without 10 mM EDTA, 50 mM dithiothreitol, or 50 mM Gly-Pro-ArgPro (Sigma). The concentrations of the applied and eluted FD1 were estimated from the absorption at 280 nm (molar absorption coefficient: 48,700 cmϪ1 MϪ1), and the relative fractions of the eluted FD1 at each pH were evaluated using the KaleidaGraph non-linear fitting program (Synergy Software)

RESULTS

DISCUSSION

Methods