Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids

Abstract

Toll-like receptors (TLRs) are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system, and its biochemical as well as ligand binding properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid (LTA) from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chain containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED. The design of novel inhibitors of TLR2, based on their ability to bind to TLR2 but not activate the TLR2 signaling pathway, may lead to the development of novel treatments for septic shock caused by Gram- positive bacteria.

The critical role of toll-like receptors — From microbial recognition to autoimmunity: A comprehensive review

“…TLR2 recognize the broadest range of ligands among TLRs due to its association with other TLRs (TLR1 and TLR6) [15-17]. Crystal structure of TLR2/TLR1 in complex with a triacyl-lipopeptide and TLR2 in complex with a diacyl-lipopeptide showed that the hydrophobic pocket of TLR2 formed by the central LRRs (LRR9 to LRR12) binds the diacylglycerol acyl chains while TLR1 interacts with the N-acyl chains of the ligands ( Figure 1A) [16].…”

“…Crystal structure of TLR2/TLR1 in complex with a triacyl-lipopeptide and TLR2 in complex with a diacyl-lipopeptide showed that the hydrophobic pocket of TLR2 formed by the central LRRs (LRR9 to LRR12) binds the diacylglycerol acyl chains while TLR1 interacts with the N-acyl chains of the ligands ( Figure 1A) [16]. Furthermore, TLR2 also recognize glycolipids such as lipoteichoic acid from Gram-positive bacteria [17, 18], lipoarabinomannan from mycobacteria [17, 19], and GPI anchor structures from Trypanosome Cruzi [20]. We have recently reported that the single hydrophobic pocket of human TLR2 ectodomain is also responsible for binding microbial glycolipids and other lipopeptides [17].…”

“…Furthermore, TLR2 also recognize glycolipids such as lipoteichoic acid from Gram-positive bacteria [17, 18], lipoarabinomannan from mycobacteria [17, 19], and GPI anchor structures from Trypanosome Cruzi [20]. We have recently reported that the single hydrophobic pocket of human TLR2 ectodomain is also responsible for binding microbial glycolipids and other lipopeptides [17]. Based on the TLR1/TLR2 and TLR2/TLR6 complex structures, homology models of hTLR10 show a ligand binding pocket similar to TLR2 [21].…”

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

“…Samples were measured at 680 nm in a CLARIOstar microplate reader (BMG Labtech). Native PAGE experiments were performed to study TLR2-ligand interactions as described (36). Purified human TLR2 (7 μM, final concentration), in some cases preincubated with SSL3ΔN or SSL3ΔN − (40 μM) for 30 min at room temperature, was mixed with Pam 2 CSK 4 Rhodamine (20 μM, InvivoGen) and incubated for 18 h at 37°C.…”

Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

“…Recently, the ligand binding properties of the recombinant human TLR2 ectodomain (hTLR2ED) were investigated (72). It turned out that the ectodomain binds synthetic bacterial and mycoplasmal lipopeptides, S. aureus lipoteichoic acid (obtained from Invivogen, San Diego, CA), and synthetic lipoarabinomannan precursors from Mycobacterium in the absence of its coreceptors TLR1 and TLR6.…”