Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function

Abstract

The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.

Figure 1.

Sequence alignments. Alignment of known dectin-1 sequences, with only the CTLDs shown. Secondary structure elements and solvent accessibilities (blue, accessible; cyan, intermediate; white, buried) of the murine protein are displayed above and below the alignments, respectively. Metal binding residues are boxed and colored yellow. Yellow triangles indicate dectin-1 residues involved in the P3₂21 asymmetric unit dimer interface, and orange triangles indicate residues mutated by Adachi et al. (2004). The alignment was formatted using ESPript (espript.ibcp.fr/ESPript/ESPript).

Figure 2.

Stereoviews showing surface characteristics of the dectin-1 monomer. (A) Two different ribbon diagram views of the dectin-1 monomer, each colored from blue at the N terminus to red at the C terminus with disulphide linkages shown as gray balls-and-sticks and the metal ion as a golden sphere. Dotted lines indicate surface regions involved in crystallographic dimers. (B) Equivalent orientations as A, showing hydrophobic patches (green) over surface representations of dectin-1. Hydrophobic patches are defined using the program GRID and are shown here as volumes of pseudo-energies contoured at –2.3 kcal mole⁻¹. The solvent accessible regions of Trp221 and His223 are represented by the yellow ellipse. (C) Equivalent orientations as A, showing the electrostatic potential surface of dectin-1 produced using the program GRASP and contoured ±20 kT (blue denotes positive; red, negative potential). The yellow ellipse indicates the solvent accessible regions of Trp221 and His223, and the positions of some of the residues and atoms responsible for the negative potential are indicated.

Figure 3.

Metal ion binding coordination in dectin-1. Ball-and-stick diagram showing the octahedral metal ion coordination in dectin-1. The distances between the calcium ion and the chelating atoms are as follows: Lys156 O, 2.3 Å; Asp158 OD2, 2.2 Å; Glu162 OE1, 2.2 Å; Glu241 OE1, 2.3 Å; HOH1, 2.2 Å; and HOH2, 2.4 Å.

Figure 4.

Biophysical studies of refolded dectin-1. (A) FACS-based assay showing β-glucan binding by refolded dectin-1 constructs. The dark gray region corresponds to dectin-1 binding, the lighter gray line indicates inhibition by glucan phosphate, and the black region corresponds to the negative control. (B) Thermal shift profiles show dectin-1 binds β-glucan and divalent cations. Measurements of dectin-1 alone (blue), with Ca²⁺/Mg²⁺ (green), with laminarin (red), and with both Ca²⁺/Mg²⁺ and laminarin (orange) are shown. The shift in melting temperature, indicated by the gray lines, reflects the increased energy required to melt the protein in the presence of the various ligands. (C) AUC measurements demonstrate that dectin-1 forms multimeric complexes in the presence of laminarin. Measurements, colored as in B, were taken at 21,000 rpm and 280 nm. In the absence of laminarin (blue and green measurements), the molecular weights suggest monomeric species, whereas in the presence of laminarin (red and orange), there is an obvious increase in molecular weight. Two possible curve fits for the measurements taken with dectin-1 plus laminarin (in red) are shown (for details, see text).

Figure 5.

Two dectin-1 monomers form a dimer into which a short β-glucan binds. (A) A cartoon diagram of the dectin-1 P3₂21 dimer, with each monomer colored from blue at the N terminus to red at the C terminus. Disulphide linkages are shown as gray balls-and-sticks, the metal ion as a golden sphere, and the bound β-glucan as yellow and red balls-and-sticks. (B) Equivalent orientation as A, showing the electrostatic potential surface of the dectin-1 P3₂21 dimer around the region where β-glucan is observed, produced using GRASP and contoured ±20 kT (blue denotes positive; red, negative potential). Bound β-glucan is shown as yellow and red balls-and-sticks. (C) Electron density for laminaritriose observed in a 2F_o–F_c composite-omit map contoured at 1σ.