Structural insight into the recognition of pathogen-derived phosphoglycolipids by C-type lectin receptor DCAR

Abstract

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca²⁺ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8-2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala¹³⁶ and Gln¹⁹⁸, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca²⁺-binding motif (Glu-Pro-Ser/EPS_168-170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Keywords: C-type lectin domain family 4 member; C-type lectins; Mycobacterium tuberculosis; X-ray crystallography; acylated phosphatidyl-myo-inositol mannoside (AcPIM); glycolipid; innate immunity; myeloid cell; pattern recognition receptor (PRR).

Figure 1.

Structure based sequence alignment of DCAR, Mincle, DCIR1, and DC-SIGN. Amino acid sequence alignment of the extracellular domains of DCAR, Mincle, DCIR1, and DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin), with identical residues highlighted in red and similar residues enclosed in blue boxes (m, mouse; c, cow; h, human). Secondary structure elements (α-helices and β-strands) of DCAR are shown above the primary structures, and each pair of cysteines involved in disulfide-bond formation are designated by a number below the sequences. The green filled circles indicate the residues participating in Ca²⁺ coordination, and the residues involved in ligand binding are enclosed in black boxes. Sequence alignment was obtained with ESPript (33).

Figure 2.

Comparison of the overall structure and Ca²⁺-binding site of DCAR and Mincle. A–C, overall structures of DCAR (A, PDB code 6KZR) and cow Mincle (B, PDB code 4KZW). The green spheres indicate the position of Ca²⁺ ions in DCAR and Mincle. Residues from the N- to C-terminal end are shown by the blue to red color gradient. The superimposed structure of DCAR (cyan) and Mincle (pink) is shown in C. D-F, the primary sugar-binding sites of DCAR (D, cyan), Mincle (E, pink), and their superimposition (F) are shown in close-up view. The spheres represent Ca²⁺ ions and Ca²⁺ coordination bonds are depicted by black dotted lines. Oxygen and nitrogen atoms are shown in red and blue, respectively.

Figure 3.

Structural comparison of the putative ligand-binding site of DCAR and Mincle. A, close-up view of the putative ligand-binding site of DCAR (cyan, left panel, PDB code 6KZR) and Mincle (pink, right panel, PDB code 4KZV) bound to glycerol (green) and trehalose (yellow), respectively. Hydrogen and Ca²⁺ coordination bonds are depicted by black dotted lines. B, superimposed structures of the residues adjacent to the putative binding site of glycerol-bound DCAR (cyan) and trehalose-bound Mincle (pink). C and D, reporter cells expressing DCAR or its mutants were stimulated with increasing amounts (30, 100, and 300 ng/well) of plate-coated Ac₂PIM2 for 18 h, and NFAT-GFP activity was assessed by flow cytometry. E, the Fc portion of human IgG (Ig), WT or mutant DCAR–Ig fusion proteins were incubated with increasing amounts (10, 30, and 100 ng/well) of plate-coated Ac₂PIM2, and binding was detected using anti-hIgG–horseradish peroxidase. The data are presented as the means ± S.D. of triplicate assays and are representative of three independent experiments (C–E).

Figure 4.

DCAR interacts with the AcPIM phosphosaccharide moiety. A, blocking of DCAR–Ig fusion protein binding to plate-coated AcPIM2 (150 ng/well) by increasing concentrations (0.0625, 0.125, 0.250, 0.5, 1, and 2 mm) of mannose and inositol-monophosphate dimannose (IPM2). The structure of both molecule is shaded in red and blue, respectively, on the structure of AcPIM2. Mannose and inositol residues are shown by Man and Ins, respectively. The data are presented as the means ± S.D. of triplicate assays and are representative of two independent experiments. B, analysis of the interaction between IPM2 and DCAR–Ig (top panels) or control Ig (lower panels) by biolayer interferometry. The fitting view is shown on the left, and the steady-state analysis is on the right.

Figure 5.

Crystal structure of the DCAR CRD in complex with IPM2. A, close-up view of the ligand-binding site bound to IPM2 (PDB code 6LFJ). Hydrogen and Ca²⁺ coordination bonds are depicted by black dotted lines, and IPM2 is in yellow. Mannose and inositol residues are shown by Man and Ins, respectively. B, stereo view of the omit map contoured at 1.9 σ level around IPM2 and shown by a blue mesh. C–E, overall structure of the ligand-free DCAR CRD (ligand-free, PDB code 6LKR) (C), the IPM2 complex (IPM2-bound) (D), and their superimposition (superimposed; root-mean-square deviation of 0.117 Å for 264 structurally equivalent Cα atoms) (E). The spheres represent Ca²⁺ ions.

Figure 6.

Contribution of a hydrophobic groove to binding with AcPIMs. A and B, overall (A) and close-up view of the primary sugar-binding site (B). Hydrophobicity is represented by a white (hydrophilic) to red (hydrophobic) gradient. The dotted oval shows the possible acyl chain–binding site, and the positions of the Ile¹³³ and 6-OH of the bound mannose are shown in white and black, respectively. The green sphere indicates the position of the Ca²⁺ ion. C and D, reporter cells expressing FcRγ only (Mock) or together with DCAR, DCAR^I133G, or DCAR^I133A were stimulated with increasing amounts (30, 100, and 300 ng/well) of plate-coated Ac₂PIM2 (C) or by coated anti-DCAR mAb (D). The data are presented as the means ± S.D. of triplicate assays and are representative of two independent experiments.