The Recognition Unit of FIBCD1 Organizes into a Noncovalently Linked Tetrameric Structure and Uses a Hydrophobic Funnel (S1) for Acetyl Group Recognition

• Published in: The Journal of biological chemistry Vol. 285; no. 2; pp. 1229 - 1238
• Main Authors: Thomsen, Theresa, Moeller, Jesper B., Schlosser, Anders, Sorensen, Grith L., Moestrup, Soren K., Palaniyar, Nades, Wallis, Russell, Mollenhauer, Jan, Holmskov, Uffe
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.01.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Binding Sites - physiology; Carbohydrate/Complex; Cattle; Cell/Endocytosis; Chitin - chemistry; Chitin - genetics; Chitin - metabolism; Humans; Immunology/Innate Immunity; Methods/Site-directed Mutagenesis; Mutagenesis, Site-Directed; Mutation, Missense; Protein Binding - physiology; Protein Structure and Folding; Protein Structure, Quaternary - physiology; Protein Structure, Tertiary - physiology; Protein/Ligand Binding; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors/Membrane; Structural Homology, Protein; Structure-Activity Relationship; Immunology/Innate Immunity; Carbohydrate/Complex; Protein/Ligand Binding; Receptors/Membrane; Cell/Endocytosis; Methods/Site-directed Mutagenesis
• DOI: 10.1074/jbc.M109.061523
• ISSN: 0021-9258

We have recently identified FIBCD1 (Fibrinogen Cdomain containing 1) as a type II transmembrane endocytic receptor located primarily in the intestinal brush border. The ectodomain of FIBCD1 comprises a coiled coil, a polycationic region, and a C-terminal FReD (fibrinogen-related domain) that assembles into disulfide-linked homotetramers. The FIBCD1-FReD binds Ca2+ dependently to acetylated structures like chitin, N-acetylated carbohydrates, and amino acids. FReDs are present in diverse innate immune pattern recognition proteins including the ficolins and horseshoe crab TL5A. Here, we use chemical cross-linking, combined with analytical ultracentrifugation and electron microscopy of the negatively stained recombinant FIBCD1-FReD to show that it assembles into noncovalent tetramers in the absence of the coiled coil. We use surface plasmon resonance, carbohydrate binding, and pulldown assays combined with site-directed mutagenesis to define the binding site involved in the interaction of FIBCD1 with acetylated structures. We show that mutations of central residues (A432V and H415G) in the hydrophobic funnel (S1) abolish the binding of FIBCD1 to acetylated bovine serum albumin and chitin. The double mutations (D393N/D395A) at the putative calcium-binding site reduce the ability of FIBCD1 to bind ligands. We conclude that the FReDs of FIBCD1 forms noncovalent tetramers and that the acetyl-binding site of FReDs of FIBCD1 is homologous to that of tachylectin 5A and M-ficolin but not to the FReD of L-ficolin. We suggest that the spatial organization of the FIBCD1-FReDs determine the molecular pattern recognition specificity and subsequent biological functions.