Mechanism for recognition of an unusual mycobacterial glycolipid by the macrophage receptor mincle

Abstract

Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1-1Glcα headgroup is liganded to a Ca(2+) in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.

Keywords: CLEC4E; Carbohydrate-binding Protein; Cord Factor; Crystal Structure; Glycobiology; Glycolipids; Mycobacterium tuberculosis.

FIGURE 1.

Organization of mincle. A, diagram of mincle showing the location of the CRD and association with the immunoreceptor tyrosine activation motif (ITAM) in the Fc receptor-γ (FcRγ) subunit through a charge-charge interaction in the membrane. B, comparison of the sequences of the predicted CRDs of bovine (Bo), human (Hu), and mouse (Mo) mincle. Conserved cysteine residues that form the three disulfide bonds characteristic of C-type CRDs are highlighted in yellow, and ligands for the conserved Ca²⁺ site are highlighted in green (7). Key residues in the secondary glucose-binding site are shaded pink, and residues that form the hydrophobic groove are shaded blue. Residues that chelate the supplemental Ca²⁺ are indicated in violet. The mouse and cow CRDs show sequence identities of 71 and 76% to the human protein, respectively.

FIGURE 2.

Binding specificity of mincle. A, SDS-PAGE of the CRD from bovine mincle expressed in E. coli and purified on trehalose-Sepharose. Refolded protein, which bound to the affinity column in the presence of Ca²⁺, was eluted with EDTA. The gel was stained with Coomassie Blue. B, glycolipid blot demonstrating bovine mincle binding to trehalose dimycolate. The blot was incubated with the biotin-tagged CRD from bovine mincle, followed by alkaline phosphate-conjugated streptavidin. C, quantitative comparison of the affinities of trehalose and glucose for mincle determined in a binding competition assay in which the biotin-tagged CRD immobilized on streptavidin-coated wells was probed with ¹²⁵I-Man₃₁-BSA. An example of one assay, performed in duplicate, is shown. The mean ratio of K_I values for α-methyl glucoside and trehalose is based on four repetitions of the assay.

FIGURE 3.

Structure of a low pH form of the mincle CRD. A, overall structure of the mincle CRD in crystals obtained at pH 5.0, in which citrate is bound at the conserved Ca²⁺ site. Ca²⁺ is shown in orange, and Na⁺ is shown in gray. The supplemental Ca²⁺-binding site, seen near the bottom of the structure, has been observed in other C-type CRDs (40). B, region of the CRD around the conserved Ca²⁺ site at which the sugar-binding site is usually located, showing a bound citrate ion. The side chain of one of the expected ligands, Asp-193, is rotated away from the Ca²⁺, and a second expected ligand, Glu-176, is in a loop encompassing Asn-170–Asp-177, which assumes an unusual conformation in which Glu-176 does not contact the conserved Ca²⁺ site. Hydrogen and coordination bonds are indicated with dotted lines, using cutoffs of 2.6 Å for coordination bonds and 3.2 Å for hydrogen bonds. C, F_o − F_c electron density for the citrate moiety, calculated by omitting the citrate from the model, contoured at 3.0 σ, and shown as green mesh. Oxygen atoms are indicated in red, and nitrogen atoms are blue.

FIGURE 4.

Structure of mincle complexed with trehalose. A, overall structure of the trehalose-CRD complex. B, F_o − F_c electron density for the trehalose saccharide, calculated by omitting the sugar residue from the model, contoured at 3.0 σ, and shown as green mesh. C, superposition of the mincle CRD bound to citrate (blue) and complexed with trehalose (green), showing the different conformation of the loop between Asn-170 and Asp-177 near the conserved Ca²⁺ site. D, arrangement of cation-binding sites in the citrate complex. A Na⁺ (gray sphere) interacts with the main chain oxygens of Leu-172 and Val-175, as well as with the side chain of Asp-177 and two water molecules. E, arrangement of cation-binding sites in the trehalose complex. In this case, the Na⁺ interacts with the main chain oxygen of Glu-176 and the carboxyl group of Asp-193, as well as the side chain of Asn-171 and two water molecules. F, primary binding site for glucose at the conserved Ca²⁺ site showing the five canonical ligands for the divalent cation, four of which also interact with the 3- and 4-OH groups of the first glucose residue. G, trehalose disaccharide interactions with both the primary and secondary binding sites. H, secondary binding site, in which Glu-135 and Arg-182 form hydrogen bonds with 2-OH of the second glucose residue. Ca²⁺ is shown in orange, Na⁺ in gray, oxygen atoms in red, and nitrogen atoms in blue. Hydrogen and coordination bonds are indicated with dashed lines, using cutoffs of 2.6 Å for coordination bonds and 3.2 Å for hydrogen bonds. van der Waals contacts, based on a 4.0-Å cutoff, are indicated with solid lines.

FIGURE 5.

pH dependence of ligand binding to mincle CRD. The binding of ¹²⁵I-Man₃₁-BSA to the biotin-tagged CRD immobilized on streptavidin-coated wells was measured.

FIGURE 6.

Mutagenesis of residues in the secondary glucose-binding site in mincle. An example of one assay, performed in duplicate, is shown. The ratios of the K_I values for α-methyl glucoside and trehalose following mutation of Glu-135 to glutamine and mutation of Arg-182 to lysine are reported as means based on four repetitions of the assay.

FIGURE 7.

Mincle interactions with glycolipid mimics. A, structures of mycolic acid and trehalose. Both X and Y are mycolic acid in trehalose dimycolate. B, enhancement of the affinity of mincle for trehalose by acylation of one or both of the 6-OH groups demonstrated in binding competition assays. Values are means ± S.D. for three to four replicate experiments.

FIGURE 8.

Hydrophobic channel adjacent to the primary binding site. A model of the trehalose octanoate conjugate was made by adjusting the O5-C5-C6-O6 dihedral angle of glucose residue 1 by 120° and forming a bond of the appropriate length with the planar carboxylate group of octanoic acid from Protein Data Base entry 1H2B. The model suggests that at least the first six carbon atoms of the acyl chain would interact with the channel. The surface of the protein is colored based on the underlying atoms: green for carbon, red for oxygen, and blue for nitrogen. The hydrocarbon chain of the octanoic acid is colored cyan.

FIGURE 9.

Mutagenesis of residues in the hydrophobic groove in mincle. Inhibition curves for the mono-octanoyl derivative of trehalose competing for binding to wild-type and mutant mincle CRDs are shown. K_I values for trehalose mono-octanoate (based on four repetitions of the assay) are indicated.

FIGURE 10.

Model of the interaction of mincle with mycobacteria. Mincle, on the surface of macrophages, is shown forming a bridge with trehalose dimycolate on the surface of mycobacteria. ITAM, immunoreceptor tyrosine activation motif; FcRγ, Fc receptor-γ subunit.