Essential Roles of Hydrophobic Residues in Both MD-2 and Toll-like Receptor 4 in Activation by Endotoxin

Abstract

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4⅐MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4⅐MD-2⅐LPS complex, driving TLR4 activation. Bacterial lipopolysaccharide (LPS)3 is recognized by the innate immune system of vertebrates via an elaborate mechanism involving the membrane receptor TLR4 (1, 2). The extracellular (or cell surface) proteins LPS-binding protein and CD14 promote extraction and transfer of individual molecules of LPS from the Gram-negative bacterial outer membrane to MD-2, either secreted monomeric soluble (s)MD-2 or MD-2 bound with high affinity to the ectodomain of TLR4 (3-7). In contrast to other Toll-like receptors, TLR4 requires an additional molecule, MD-2, for ligand recognition (8). In contrast to MD-2, there has been no evidence of direct binding of LPS to TLR4 (9, 10). Although LPS, and particularly the lipid A portion of LPS, is generally conserved among Gram-negative bacteria, there are many variables in LPS structure that affect TLR4 activation. Most important is the acylation pattern of the lipid A moiety, which represents the minimal segment of LPS that can trigger activation of TLR4 (11). Comparison of crystal structures of MD-2 with and without bound tetraacylated lipid IVa indicates no significant alteration of the protein fold in the absence or presence of bound ligand (12). It has been proposed that both LPS and MD-2 are key to the different effects of tetraversus hexaacylated LPS on TLR4 (8,13,14). Lipid IVa complexed to murine MD-2 has weak agonist effects on murine TLR4 but acts as a receptor antagonist in the same complex containing human MD-2. Hexaacylated endotoxins complexed to human or murin...

MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA

“…9). Leu-125 sits on the Phe-126 loop, which is essential for receptor dimerization (13,14). However, Phe-126 does not directly bridge dimerization in the hTLR4⅐hMD-2⅐LPS co-crystal structure (12); it actually folds back toward hMD-2 to accommodate the extra acyl chain of LPS that lies on hMD-2 surface in the co-crystal structure (12).…”

“…2a, only the upper portion of the fourth acyl chain on lipid IV A was expected to be involved in hydrophobic interactions with mMD-2 and mTLR4 at the dimerization interface. In comparison, hydrophobic interactions between mMD-2 and mTLR4 at the dimerization interface in the mTLR4⅐mMD-2⅐lipid IV A model were expected to be similar to those in the hTLR4⅐hMD-2⅐LPS co-crystal structure (12), because virtually all hydrophobic residues on MD-2 and TLR4 essential for receptor dimerization (13,14) are conserved between the two species (12). In addition, mMD-2 has the hydrophobic Leu-125 in place of Lys-125 in hMD-2 ( Fig.…”

“…Surprisingly, the LPS core polysaccharide, which is not required to induce a proinflammatory response, also interacts extensively with TLR4 in the cocrystal structure. Studies with site-directed mutagenesis con-firmed that Phe-126 on human MD-2 (hMD-2) is essential for receptor dimerization (13) and demonstrated that hydrophobic residues on hMD-2 and human TLR4 (hTLR4) are required for receptor activation (14).…”

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

“…Recently determined crystal structure of the TLR4⅐MD-2-RaLPS complex and a model, based on mutations of MD-2 and TLR4 demonstrated the occupation of the complete pocket of MD-2 with acyl chains of the LPS. Therefore, even a small ligand bound to its thiol group could (43,44) interfere with lipid A binding, particularly with an agonistic chemotype, that typically contains six acyl chains. Acrolein has been reported to inhibit the NF-B activation by endotoxin (45), and it has recently been shown that it suppresses the LPS-induced TLR4 dimerization upstream of MyD88 (46).…”

The role of protein–protein interactions in Toll-like receptor function

“…This is due to the nature of its main PAMP, lipopolysaccharide (LPS), a glycolipid from Gramnegative bacterial outer membranes [12,[47][48][49][50][51][52][53][54]. Only small amounts of LPS are required to stimulate the TLR4 pathway and hence alert the immune system to invading pathogens, and overstimulation of this pathway can lead to sepsis.…”