Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL Atsushi Furukawaa,b,1, Jun Kamishikiryoc,1, Daiki Morid,1, Kenji Toyonagad, Yuki Okabea,2, Aya Tojia, Ryo Kandaa, Yasunobu Miyaked, Toyoyuki Osea, Sho Yamasakid,3, and Katsumi Maenakaa,b,3 aLaboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan; bCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama 332-0012, Japan; cFaculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama 729-0292, Japan; and dDivision of Molecular Immunology, Research Center for Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan Edited* by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved September 9, 2013 (received for review July 7, 2013) Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; motif (ITAM) in the FcRγ chain, which provides a binding site CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are recep- for the Syk tyrosine kinase. Syk activates the caspase recruit- tors for the cord factor TDM (trehalose-6,6′-dimycolate), a unique ment domain family member 9-mediated NF-κB signaling glycolipid of mycobacterial cell-surface components, and activate pathway to promote the expression of TNF and IL-6. A recent immune cells to confer adjuvant activity. Although it is known that report revealed that Mincle plays a nonredundant role in T-cell receptor–TDM interactions require both sugar and lipid moieties of immune responses to infection by microbes and in the adju- TDM, the mechanisms of glycolipid recognition by Mincle and MCL vanticity of mycobacterial cord factor and its synthetic analog, remain unclear. We here report the crystal structures of Mincle, trehalose-dibehenate (14, 15). MCL, and the Mincle–citric acid complex. The structures revealed Macrophage C-type lectin (MCL; also called CLEC4D) is another C-type lectin receptor expressed in myeloid cells (16, that these receptors are capable of interacting with sugar in γ 2+ 17). Recently, we found that MCL is also an FcR -coupled ac- aCa -dependent manner, as observed in other C-type lectins. tivating receptor that binds to TDM (15). MCL is distinct from However, Mincle and MCL uniquely possess shallow hydrophobic Mincle in the following ways: (i) The expression of Mincle is regions found adjacent to their putative sugar binding sites, which inducible, whereas MCL is constitutively expressed in myeloid reasonably locate for recognition of fatty acid moieties of glyco- cells; (ii) MCL shows weaker binding affinity to TDM than does IMMUNOLOGY lipids. Functional studies using mutant receptors as well as glyco- Mincle; and (iii) the glutamic acid-proline-asparagine (EPN) lipid ligands support this deduced binding mode. These results motif, a typical glucose/mannose-binding motif, is conserved in give insight into the molecular mechanism of glycolipid recogni- Mincle but not in MCL. tion through C-type lectin receptors, which may provide clues to We now report the crystal structures of Mincle and MCL, as rational design for effective adjuvants. well as Mincle complexed with citric acid. They have similar overall structures to other typical CLRs, but exhibit character- 2+ X-ray crystallography | innate immunity | mycobacteria | istic conformations in the vicinity of the Ca -binding motif. A pattern-recognition receptors | myeloid cells patch of hydrophobic amino acids located adjacent to the car- bohydrate binding site may likely contribute to the recognition of attern-recognition receptors (PRRs) play important roles in fi Pinnate immunity. PRRs recognize nucleotides, sugars, lip- Signi cance opolysaccharides, other pathogen components, and self-ligands, and consequently trigger intracellular signaling cascades that Here we report the crystal structures of human C-type lectin initiate innate and adaptive immune responses (1). Among them, receptors Mincle (macrophage inducible C-type “calcium- Toll-like receptors (TLRs) are well-characterized receptors, in dependent” lectin; CLEC4E) and MCL (macrophage C-type lec- terms of their ligand specificities, ligand-recognition mecha- tin; CLEC4D), both of which are receptors for mycobacterial nisms, and signaling pathways (2–4). C-type lectin receptors glycolipid adjuvant cord factor (also called trehalose-6,6′- (CLRs) are also a large family of PRRs (5–7). The term “C-type dimycolate; TDM). Our structural and functional studies clearly + lectin” was introduced to distinguish a group of Ca2 -dependent reveal the simultaneous recognition of sugar and lipid moieties lectins from other lectins. In the CLRs, two amino acids har- by Mincle and MCL, distinct from other C-type lectin receptors. boring long carbonyl side chains separated by a proline in a cis Because better adjuvants are desired for enhancing vaccination + conformation coordinate a Ca2 ion, which forms hydrogen effects of medical treatments for infectious diseases, cancer, bonds with monosaccharides and determines the binding speci- and so forth, these structures provide a framework for the ficity. CLRs have broad recognition abilities toward not only rational design of more effective adjuvants than TDM. saccharides but also proteins (5, 7–9). For instance, human NKR- Author contributions: A.F., J.K., D.M., S.Y., and K.M. designed research; A.F., J.K., D.M., P1 interacts with Lectin-like transcript 1, and some members of K.T., Y.O., A.T., R.K., Y.M., and T.O. performed research; A.F., J.K., D.M., K.T., Y.O., R.K., the CD94/NKG2 family interact with HLA-E. Y.M., T.O., S.Y., and K.M. analyzed data; and A.F., J.K., D.M., T.O., S.Y., and K.M. wrote Macrophage inducible C-type lectin (Mincle; also called the paper. CLEC4E) is a type II transmembrane C-type lectin receptor that The authors declare no conflict of interest. is expressed in macrophages, dendritic cells, and monocytes *This Direct Submission article had a prearranged editor. γ upon stimulation (10). We have reported that Mincle is an FcR - Data deposition: The crystallography, atomic coordinates, and structure factors reported coupled activating receptor that recognizes pathogenic fungi and in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes mycobacteria (11–13). Detailed investigations of the ligands of 3WH3, 3WH2, and 3WHD). Mincle revealed that Mincle binds glycolipids, such as trehalose- 1A.F., J.K., and D.M. contributed equally to this work. ′ Mycobacterium tuberculosi 6,6 -dimycolate (TDM) from s, and 2Present address: Department of Functional Biological Chemistry, Division of Science, novel glyceroglycolipids from Malassezia fungus. Malassezia and Fukuoka University, Fukuoka 814-0180, Japan. M. tuberculosi s ligands are recognized through the carbohydrate- 3To whom correspondence may be addressed. E-mail: [email protected] or recognition domain (CRD) in the extracellular region of Mincle [email protected]. (11, 12). The binding of TDM to Mincle leads to the phos- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. phorylation of the immunoreceptor tyrosine-based activation 1073/pnas.1312649110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1312649110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 the fatty acid chain of TDM. The mutational analysis essentially α-helices (α1andα2) and 11 β-strands (β1–β11) (Fig. 2A), which supports this TDM-binding model, and may also explain the is a typical structural organization of CLRs and partly similar different affinities of MCL and Mincle. to the solution structure of MCL registered in the Protein Data Bank (PDB) (ID code 2LS8)(Fig. S1E). Two MCL molecules Results exist in the asymmetric unit. Gel-filtration analysis showed a Preparation, Crystallization, and Structural Determination of MCL. mixture of the peaks (Fig. S1A), suggesting that MCL may have The extracellular domain of human MCL (residues 61–215) some conformational variation. (Fig. 1) was expressed in Escherichia coli as inclusion bodies, and + was refolded in vitro by a dilution method. Ca2 ions were re- Preparation, Crystallization, and Structural Determination of Mincle. quired in the refolding procedure, and the crude, refolded MCL Using a similar refolding method to that for MCL, we also was purified by sequential gel-filtration chromatography steps prepared the extracellular domain of Mincle (residues 74–219) (Fig. S1 A and C). The purified MCL was crystallized by the (Fig. 1). The expression, refolding, and purification were suc- hanging-drop method with 0.1 M bis-Tris propane (pH 6.5), 0.2 M cessful. However, the crystallization was not successful, because potassium thiocyanate, and 20% (wt/wt) PEG 3350. Crystals of the the refolded Mincle was not sufficiently soluble at high concen- MCL protein [the space group was I-centered orthorhombic trations. To improve the protein solubility, we performed site- (I222), and the unit-cell parameters were a = 85.19 Å, b = 96.06 Å, directed mutagenesis and changed the hydrophobic amino acids c = 104.53 Å] were obtained, and the dataset was collected to the presumably located on the surface of the Mincle protein to hy- resolution limit of 2.2 Å at the BL32XU beamline at SPring-8 drophilic amino acids, as found in the corresponding residues (Harima, Japan) (Table S1). The crystal structure of MCL has 2 of MCL. Among them, the mutant with the substitution of iso- leucine to lysine at residue 99 (I99K mutant) formed good crystals by the hanging-drop method under two conditions. One is 1 M lithium chloride, 0.1 M citric acid (pH 4), and 20% (wt/vol) PEG 6000, and the other is 0.2 M NH4SO4, 0.1 M bis-Tris (pH 5.5), 25% (wt/vol) PEG 3350. These diffraction data were col- lected to the resolution limit of 1.3 and 1.35 Å at the BL5A and BL17A beamlines at High Energy Accelerator Research Orga- nization (KEK) (Tsukuba, Japan), respectively.