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

Abstract

Lipopolysaccharide (LPS), also known as endotoxin, activates the innate immune response through toll-like receptor 4 (TLR4) and its coreceptor, MD-2. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. Tetraacylated lipid IVa, a synthetic lipid A precursor, acts as a weak agonist to mouse TLR4/MD-2, but as an antagonist to human TLR4/MD-2. However, it remains unclear as to how LPS and lipid IVa show agonistic or antagonistic activities in a species-specific manner. The present study reports the crystal structures of mouse TLR4/MD-2/LPS and TLR4/MD-2/lipid IVa complexes at 2.5 and 2.7 Å resolutions, respectively. Mouse TLR4/MD-2/LPS exhibited an agonistic "m"-shaped 2:2:2 complex similar to the human TLR4/MD-2/LPS complex. Mouse TLR4/MD-2/lipid IVa complex also showed an agonistic structural feature, exhibiting architecture similar to the 2:2:2 complex. Remarkably, lipid IVa in the mouse TLR4/MD-2 complex occupied nearly the same space as LPS, although lipid IVa lacked the two acyl chains. Human MD-2 binds lipid IVa in an antagonistic manner completely differently from the way mouse MD-2 does. Together, the results provide structural evidence of the agonistic property of lipid IVa on mouse TLR4/MD-2 and deepen understanding of the ligand binding and dimerization mechanism by the structurally diverse LPS variants.

Tetraacylated Lipid A and Paclitaxel-Selective Activation of TLR4/MD-2 Conferred through Hydrophobic Interactions

“…This finding suggests that interactions within the mTLR4/MD-2/LPS activation complex are stronger; therefore, deletion of a contribution of a single phenylalanine residue is not sufficient to prevent the receptor dimerization. This is consistent with evidence from the crystal structures of TLR4/MD-2/LPS complexes that indicate higher numbers of interactions sites within the dimerization interface of mouse in comparison with hTLR4/MD-2/LPS complexes (5,12). In the recently determined crystal structure of the mouse receptor complex TLR4/MD-2 with lipid IVa and LPS, both ligands adopted very similar conformations, with partial solvent exposure of the R2 chain of the lipid IVa or lipid A, allowing its interaction with the secondary binding site of the TLR4 ECD (12).…”

“…This is consistent with evidence from the crystal structures of TLR4/MD-2/LPS complexes that indicate higher numbers of interactions sites within the dimerization interface of mouse in comparison with hTLR4/MD-2/LPS complexes (5,12). In the recently determined crystal structure of the mouse receptor complex TLR4/MD-2 with lipid IVa and LPS, both ligands adopted very similar conformations, with partial solvent exposure of the R2 chain of the lipid IVa or lipid A, allowing its interaction with the secondary binding site of the TLR4 ECD (12). F461 interacts in both complexes with the hydrophobic loop of MD-2, comprising residues at positions 82, 85, and 87, which have to remain hydrophobic to support activation as we have shown before (7).…”

“…The reason for antagonism of lipid IVa in case of F438A or F461A mutants is that both of the phenylalanine residues are required to stabilize the dimeric complex with lipid IVa. Weaker lipid IVa-mediated complex is supported by the absence of dimerization of mTLR4 ECD /MD-2/lipid IVa in the solution, whereas dimers are formed in the crystal and at the cell membrane (12).…”

“…However, a role for hydrophobic interactions at the dimerization interface of TLR4/ MD-2/lipid IVa complexes has recently been supported by the crystal structure of murine TLR4/MD-2/lipid IVa (12), as the lipid IVa has a very similar arrangement of the acyl chains as the hexaacylated lipid A. Because paclitaxel contains no charged groups, a role for electrostatic interactions in activation of mTLR4/MD-2 by paclitaxel seems unlikely (13).…”

“…1) (11). Comparison of crystal structures of mouse and hMD-2 (6 TLR4 ectodomain [TLR4 ECD ] and/or bound ligand) have demonstrated discrete ligand-dependent conformational changes in MD-2 that correlate with the TLR4 agonist properties of the ligand/MD-2 complex (5,12). When hexaacylated lipid A or LPS is bound to hMD-2, the aromatic side chain of F126 is shifted toward the hydrophobic pocket of MD-2.…”