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/EPS168-170). 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.
Highlights
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
Dendritic cell immunoactivating receptor (DCAR, or Clec4b1) is a CLR expressed by myeloid cells that was originally described by high sequence similarity with dendritic cell immunoreceptor 1 (DCIR1, or Clec4a2) in mouse [6]
We recently reported that DCAR functions as a receptor for mycobacteria through the recognition of unique phosphoglycolipids abundantly found in mycobacterial envelopes, called acylated phosphatidyl-myo-inositol mannosides (AcPIMs) [8]
Summary
The CRD (residues 78 –209) of murine DCAR was expressed as inclusion bodies in Escherichia coli and refolded in vitro by a dilution method. DCAR CRD crystals were obtained by the sitting-drop method, the structure was solved at a resolution of 2.3 Å, and the asymmetric unit contained two DCAR molecules (Table 1). The structure of DCAR is composed of two flanking antiparallel ␣-helices (␣1 and ␣2) and nine -strands (1 to 9) and is stabilized by three disulfide bonds similar to other CLR family members (Fig. 1). These results show that DCAR has an overall typical C-type lectin domain fold
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