Crystal structure of the tetrameric fibrinogen-like recognition domain of fibrinogen C domain containing 1 (FIBCD1) protein

Abstract

The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn(340) N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.

Keywords: Acetyl Binding; Chitin Receptor; Crystal Structure; FIBCD1; Fibrinogen-like Domain; Ligand-binding Protein; Pattern Recognition Receptor; Receptor Structure-Function; Structural Biology.

FIGURE 1.

Alignment and sequence homology (identity) of the fibrinogen-like domains of FIBCD1, TL5A, L-ficolin, M-ficolin, and H-ficolin based on structural superposition. Sequence numbers and secondary structure elements on the top refer to the FIBCD1 sequence with the numbering of the helices and strands based on the secondary structure elements assigned in TL5A and L-ficolin. The loops L1, L2, and L3 (see “Results”) are indicated. The S1 and calcium binding site residues are highlighted in green (S1) and yellow respectively, with the S3 binding site highlighted in gray. Residues that bind the additional sulfate in proximity to S1 are boxed.

FIGURE 2.

Homotetrameric structure of the recognition domains of FIBCD1. a, subunit A tetrameric native structure of FIBCD1 illustrating the crystal contact, mediated through the N-linked glycan, with the subunit B tetramer (one protomer shown in green). The four binding sites S1–S4 are labeled. The key amino acids His²⁶⁴ and Val³⁵⁷ at the protomer-protomer interface in loops L1 and L2, respectively, are shown as stick models. b, overlay of the FIBCD1 and TL5A tetramers showing the relative orientation of the protomers within the tetrameric molecule.

FIGURE 3.

Acetyl binding site S1 in each protomer of the subunit A tetramer of the native FIBCD1 structure. The acetate and sulfate ions located in and in proximity to the S1 acetyl binding pocket are shown. a, key interacting amino acids. b, charged surface representation of the extended S1 site including the acetyl binding pocket and the adjacent pocket which accommodates a sulfate ion.

FIGURE 4.

Acetyl binding site S1 in FIBCD1 showing the key amino acids and interactions between bound ligand and protein. a, native FIBCD1 subunit A showing the acetate and sulfate ions. b, native FIBCD1 subunit B showing the Asn³⁴⁰ glycan GlcNAc from the subunit A tetramer inserted in to the acetyl binding pocket. c, subunit B of the ManNAc-bound structure showing the bound ManNAc and the displaced subunit A glycan.

FIGURE 5.

Acetyl binding site S1 in each protomer of the subunit B tetramer of the native FIBCD1 structure. The Asn³⁴⁰ glycan GlcNAc from the subunit A tetramer inserts in to the acetyl binding pocket S1 of subunit B. a, structure of the binding site and the bound glycan. b, 2F_o − F_c electron density contoured at 2σ.

FIGURE 6.

Acetyl binding site S1 in the ManNAc-bound FIBCD1 structure. a and b, binding site in each protomer of the subunit A tetramer. c, binding site in each protomer of the subunit B tetramer where the N-linked GlcNAc from the subunit A tetramer in the native structure is displaced by ManNAc.

FIGURE 7.

Orthogonal views of the overlaid bound ligands in the FIBCD1 S1 acetyl binding site generated by superposing (least squares fit of the main chain atoms) subunits A and B in both the ManNAc-bound structure and the native structure. Ligands shown are ManNAc in the subunit A tetramer of the ManNAc-bound structure (yellow), the N-linked glycan GlcNAc from the subunit A tetramer bound in the native subunit B tetramer (orange), the acetate ion in the subunit A tetramer of the native structure (green), and ManNAc in the subunit B tetramer of the ManNAc bound structure (cyan).