MD-2-mediated ionic interactions between lipid A and TLR4 are essential for receptor activation.

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Lipopolysaccharide (LPS) triggers innate immunity via Toll-like receptor 4 (TLR4) and MD-2.
• Lipid A, the endotoxic part of LPS, activates myeloid cells, but the mechanism is unclear.
• Lipid IV(A), a lipid A precursor, shows species-specific activity, activating rodent cells while inhibiting human cells.

Purpose of the Study:

• To elucidate the molecular basis for the species-specific activation of TLR4 by lipid IV(A).
• To investigate the role of ionic interactions in the dimerization and activation of TLR4 complexes.

Main Methods:

• Utilized stable TLR4-expressing cell lines and MD-2-deficient macrophages.
• Employed computational studies to analyze molecular interactions.
• Performed site-directed mutagenesis on TLR4 residues.

Main Results:

• Mouse TLR4 and MD-2 are essential for lipid IV(A) activation.
• Specific ionic interactions between lipid IV(A)'s 4'-phosphate and positively charged residues (Lys367, Arg434) on mouse TLR4 were identified at the dimerization interface.
• Mutating these residues in mouse TLR4 abolished lipid IV(A) response, while enabling these interactions in human TLR4 conferred responsiveness.

Conclusions:

• Ionic interactions at the TLR4 dimerization interface are critical for lipid IV(A) species specificity.
• This study decouples receptor engagement from dimerization, highlighting the functional role of ionic interactions in TLR4 activation.
• Lipid IV(A) serves as a selective agonist to dissect TLR4 signaling pathways.