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

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 [13C]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 Phe126 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 Phe126 is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe126 may act as a “hydrophobic switch,” driving agonist-dependent contacts needed for TLR4 dimerization and activation. Phenylalanine 126 of MD-2 is essential for endotoxin-induced TLR4 activation. NMR of 13C-labeled endotoxin bound to wt or F126A MD-2 ± TLR4 ectodomain reveals effects of Phe126 on interactions of MD-2 ± TLR4 with endotoxin. Phe126 acts as a “hydrophobic switch” promoting agonist-dependent TLR4 dimerization. This study describes a novel approach to further define the structural requirements for endotoxin-induced TLR4 activation.