Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle

Abstract

The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages.

Intracellular metabolite β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity

“…A common Mincle ligand signature structure has been predicted based on a number of identified ligands (6,7,(33)(34)(35) in combination with the Mincle protein structure (9)(10)(11)(12). A polar head consisting of glucose or mannose and a hydrophobic chain appear to be the minimum requirement for ligand activity.…”

“…All six β-GlcCer species examined in this study satisfy these criteria as they harbor a polar glucose head and two acyl chains within the ceramide moiety. It remains unclear why the unsaturated β-GlcCer C24:1(15Z) possesses the most potent ligand activity, as the crystal structure of Mincle (9,10,12) suggested that the double bond of C24:1 is likely to be located away from the Mincle interacting site. Cocrystallization of Mincle protein and β-GlcCer C24:1 should clarify this issue.…”

“…In addition, Mincle recognizes pathogens, including mycobacteria through bipolar glycolipids, such as trehalose 6,6′-dimycolate (TDM), which act as pathogen-associated molecular patterns (6)(7)(8). Structural analysis of the Mincle protein has revealed how its unique ligandbinding site is suitable for glycolipid recognition (9)(10)(11)(12). Hence, we hypothesized that Mincle may recognize endogenous glycolipids released from damaged cells.…”

Rational design of adjuvants targeting the C-type lectin Mincle

“…Taken together, structural, binding, and mutagenesis data support a model for TDM and TDB binding in which the two glucose units and also, surprisingly, one acyl chain interact directly with the CRD of Mincle (8,13,15). The precise molecular mechanisms of the glycolipid-Mincle interaction are far from being completely understood, particularly with respect to how the two complex MAs of TDM are recognized by the receptor.…”

“…These structural regions were previously found to adopt different conformations in the crystallized forms of human Mincle (14) compared with the bovine Mincle (13). Moreover, it has been suggested that the conformation of the loop between residues N170 and D177 could play a key role in the ability of Mincle to bind to the glycolipid (15,25).…”