Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Abstract

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

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

“…Lipid IV A is a partial agonist in equine cells (19). However, similar to human MD-2, equine MD-2 has the positively charged Arg-122 at the hydrophobic pocket entrance.…”

“…Human MD-2 has positively charged Lys-125 at the dimerization interface close to the pocket entrance, whereas mMD-2 has hydrophobic Leu-125 at this position. A little further away from the pocket entrance, hMD-2 has positively charged Lys-58 on the ␤4 strand that has been implicated in lipid IV A discrimination (19), whereas mMD-2 has negatively charged Asn-58 at this position. Because the acyl chains of LPS bind to the MD-2 hydrophobic pocket, and the diglucosamine backbone of LPS sits next to the pocket entrance (12,13), modifications of MD-2 surface charges at residues 122, 125, and 58 likely affect lipid binding and directly influence lipid IV A species specificity.…”

Structural basis of species-specific endotoxin sensing by innate immune receptor TLR4/MD-2

“…In particular, it acts as a weak agonist on mouse and as an antagonist on human cells (20,21). Although several studies have investigated the species-specific activity of lipid IVa (22)(23)(24)(25)(26)(27)(28), these studies primarily used mutational and computational simulation methods. Structural information on the agonistic form of TLR4/MD-2 is limited to the hTLR4/MD-2/LPS complex; no structures of mTLR4/MD-2 complexed with LPS or lipid IVa are currently available.…”

Toll-like Receptor Activation of Human Cells by Synthetic Triacylated Lipid A-like Molecules

“…These results are in accordance with studies showing that different lipid A-derived structures can be TLR4/MD-2 agonists or antagonists and others TLR2 agonists. The critical factors for their agonistic versus antagonistic effects and their promiscuity for TLR2 versus TLR4 are the number of acyl chains, the length of the carbon chains, the number and the distribution of negative charges, the asymmetry of the acyl chains, and the species of TLR4 (5)(6)(7)(8). It has been shown that lipid IVa, for example, can be an agonist in mouse and horse TLR4/MD-2 but is clearly an LPS antagonist in human TLR4/ MD-2 (8,20).…”

“…This is particularly true for TLR2 and TLR4, which have been crystallized with their respective ligands (2,4). Individual LPS molecules may act as agonists in some mammalian species and as antagonists in others (5)(6)(7)(8). As an example, monomerized LPS binds CD14 and is then transferred onto MD-2 and TLR4.…”