NMR Studies of Hexaacylated Endotoxin Bound to Wild-type and F126A Mutant MD-2 and MD-2·TLR4 Ectodomain Complexes

Abstract

Host response to invasion by many gram-negative bacteria depends upon activation of Toll-like receptor 4 (TLR4) by endotoxin presented as a monomer bound to myeloid differentiation factor 2 (MD-2). Metabolic labeling of hexaacylated endotoxin (LOS) from Neisseria meningitidis with [(13)C]acetate allowed the use of NMR to examine structural properties of the fatty acyl chains of LOS present in TLR4-agonistic and -antagonistic binary and ternary complexes with, respectively, wild-type or mutant (F126A) MD-2 ± TLR4 ectodomain. Chemical shift perturbation indicates that Phe(126) affects the environment and/or position of each of the bound fatty acyl chains both in the binary LOS·MD-2 complex and in the ternary LOS·MD-2·TLR4 ectodomain complex. In both wild-type and mutant LOS·MD-2 complexes, one of the six fatty acyl chains of LOS is more susceptible to paramagnetic attenuation, suggesting protrusion of that fatty acyl chain from the hydrophobic pocket of MD-2, independent of association with TLR4. These findings indicate that re-orientation of the aromatic side chain of Phe(126) is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe(126) may act as a "hydrophobic switch," driving agonist-dependent contacts needed for TLR4 dimerization and activation.

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor

“…NMR analysis combined with metabolic labeling of a hexaacylated agonist recently provided support for the idea that Phe-126 acts as a "hydrophobic switch" (16). In both MD-2 and its F126A mutant, a single fatty acyl chain was shown to be more susceptible to paramagnetic attenuation, independent of TLR4 association, whereas the local environment of bound lipid tails was altered in the F126A mutant compared with wild type, suggesting that Phe-126 may be important in promoting formation of the active [TLR4⅐MD-2] 2 complex (16).…”

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.…”

“…(LBP) and CD14, but the binding of agonistic LPS to MD-2 is the key factor in determining TLR4 signaling, since MD-2 binds to the amino-terminal region of TLR4 and mediates active complex formation [48][49][50][51][52][53][59][60][61].…”

“…Thus, in the presence of a bound agonist, F126 remains within the MD-2 cavity (Fig 3A), enabling MD-2 residues including I80, V82, and L87 and an exposed lipid tail to form extensive hydrophobic contacts with TLR4' residues at the secondary dimerization interface; these include F440', L444', and F463', supported by a salt bridge between E422' and K125 on MD-2 [48,57]. However, in the presence of antagonists or in the ligand-free state, F126 was observed to spontaneously reorient and exit the MD-2 cavity, leading to a disruption of the hydrophobic cluster at the secondary dimerization interface and a loss of as much as ~400 Å 2 of buried surface area between TLR4' and MD-2 [48,53] (Fig 3B). Over longer timescales, it is expected that such disruption would lead to a loss of TLR4 dimerization and consequently separation of the TIR juxtaposition necessary for downstream signaling.…”

“…Indeed, analysis of the dominant simulated motions associated with loss of agonist indicates that the carboxy-termini of the TLR4 ectodomains exhibit lateral fluctuations associated with chain separation (Fig 2C, D), not unlike the conformational changes observed in crystal structures of the pre-formed TLR8 dimer upon ligand unbinding (Fig 2A, B). The idea that F126 acts as a molecular switch to determine the key protein-protein interface for TLR4 dimerization is supported by mutational studies, which reveal that F126 and nearby residues in this loop do not prevent lipid ligand binding but interfere with signaling [12,48,53,57], and by NMR studies using metabolically labeled endotoxin which demonstrated that this loop reorients upon ligand binding [53].…”