Trivalent Recognition Unit of Innate Immunity System

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 1.9 Å resolution. Although the FD1 monomer shares a common fold with the fibrinogen ␥ 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.Surveillance systems of innate immunity are present in all multicellular organisms and play a crucial role in the first line of defense against pathogens. Ficolins, as well as collectins, are one of the most important groups of pattern recognition molecules in the innate immunity systems (1-7) and have been identified in both vertebrates and invertebrates (6). Ficolins are comprised of a collagen-like domain at the N terminus and a fibrinogen-like domain (FBG), 3 which is the sugar-binding site, at the C terminus (8, 9). Collectins, such as mannose-binding lectin (MBL), lung surfactant protein A, and surfactant protein D, also 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 Ca 2ϩ dependently. The CRD on MBL, surfactant protein A, and surfactant protein D forms a trimeric structure through a triple ␣-helical coiled-coil at a short neck region between the collagen-like domain and the CRD (10 -12). Ficolins also form trimers (8,13,14), although the mechanism of trimerization is unclear. Both ficolins and collectins form trimer-based multimers that are N-terminally linked by disulfide bonds (15). Ficolins and MBL also interact with MBL-associated serine proteases, and their complexes activate the lectin complement pathway (6, 16 -23).Ficolins were originally discovered in porcine uterus membrane extracts as transforming growth factor-␤-binding proteins (24,25). In human, L-ficolin and H-ficolin in serum and M-ficolin in cells have been characterized (9, 14, 26 -29). Lficolin (synonymous with ficolin-2 or Ficolin/P35) binds to GlcNAc (29, 30) and GalNAc (8). The binding ability is inhibited by acetylated c...

Trimeric structure and conformational equilibrium of M-ficolin fibrinogen-like domain

“…The amino acid sequence of FD1 is 49% and 45% identical to that of the FBG domain of tachylectin-5A and the human fibrinogen fragment, respectively. The crystal structure revealed that the hydrophobic side-chains of Phe127 and Leu128 are important for trimer formation by FD1 (Tanio et al, 2007). Indeed, the replacement of Phe127 with Thr in FD1 drastically decreased the monomer-monomer interaction in solution, as judged by a dynamic light-scattering experiment (data not shown).…”

“…2a). The ligand-binding study of FD1 in solution revealed that the ligand-binding site is located near the Ca 2+ binding site, the Cys270-Cys283 disulfide bond, and an unidentified group with a pK a of 6.2 (Tanio et al, 2007). The group with a pK a of 6.2 was tentatively assigned to His284.…”

“…The crystal structure of FD1 was solved by molecular replacement using the crystal structure of tachylectin-5A (PDB code 1jc9; Kairies et al, 2001). The structure contains one trimeric form of FD1 in the asymmetric unit (Tanio et al, 2007). The overall structure of the FD1 monomer is similar to that of tachylectin-5A (Kairies et al, 2001) and the human fibrinogen fragment Yee et al, 1997), and consists of three domains (A, B and P; Fig.…”

“…We previously developed a model describing the conformational equilibrium at the P domain between the active (A) and non-active (N) states at any pH, from an analysis of the pH-dependent GlcNAcbinding activity of FD1 in solution (Tanio et al, 2007). In this model the equilibrium depends on the state of a group with a pK a of 6.2, which is probably the His284 side-chain.…”

“…Interestingly, although the sugars recognized by ficolins also exist on the surface of the host cell, the proteins can discriminate between the host cell (self) and pathogens (non-self). To investigate the detailed mechanism of discrimination between self and non-self by ficolins, we have determined the crystal structure of the human M-ficolin FBG domain (FD1) at 1.9 Å resolution, using synchrotron radiation at beamline BL24XU at the SPring-8 facility in Japan (PDB code 2d39; Tanio et al, 2006Tanio et al, , 2007. The crystal structure of FD1, together with its pHdependent ligand binding activity, provides insight into the discrimination mechanism by ficolins.…”