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.
Highlights
The extracellular domain of mincle contains a C-type carbohydrate recognition domain (CRD).3 Previous structural studies have revealed that the CRD has many of the features seen in other modules of this type, including a primary sugar-binding site centered on a conserved Ca2ϩ that binds to one of the two glucose residues that are linked ␣1,1 in trehalose [7]
Despite the insights that have been obtained into cow, human, and mouse mincle structures, several aspects of the structure of the CRD and the way it interacts with ligands could not be deduced from the previous analysis
Other structurally distinct ligands are based on the natural product brartemicin in which modified forms of benzoic acid are esterified to the glucose residues [12]
Summary
Effect of 6-OH Substituents on Binding to Mincle—In the mycobacterial glycolipid trehalose dimycolate, the 6-OH groups of both glucose residues of trehalose are esterified to -branched fatty acids. In one class of ligands, the long mycolic acid chains have been replaced by shorter, linear fatty acids [7, 10] These compounds are synthetically accessible using a lipase under non-aqueous conditions. Despite the variation in the structures of the acylated groups, the general pattern that emerges from these studies is that binding is enhanced by increasing the size of the groups attached to trehalose To investigate this relationship in more detail, the range of the linear acyl groups tested with bovine mincle was extended to include diacylated ligands bearing 5- and 6-carbon linear fatty acids and monoacylated ligands with 7-, 8-, 10-, and 12-carbon linear fatty acids. Results are reported as means Ϯ S.D. for n ϭ 3– 4 separate experiments, each performed in duplicate
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