Journal of Biological Chemistry
Volume 285, Issue 36, 3 September 2010, Pages 27935-27943
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Signal Transduction
MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA*

https://doi.org/10.1074/jbc.M110.134668Get rights and content
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Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)·MD-2 complex. A synthetic lipid A precursor, lipid IVA, induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IVA in mice, with TLR4 interacting through specific surface charges at the dimerization interface. In this study, we used site-directed mutagenesis to identify the MD-2 residues that determine lipid IVA species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IVA. Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IVA, effectively converting the murine cellular response to a human-like response. In human cells, however, simultaneous mutations of K122E, K125L, Y41F, and R69G on human MD-2 were required to promote a response to lipid IVA. Combining the human MD-2 quadruple mutations with the human TLR4 E369K/Q436R mutations completely converted the human MD-2/human TLR4 receptor to a murine-like receptor. Because MD-2 residues 122 and 125 reside at the dimerization interface near the pocket entrance, surface charge differences here directly affect receptor dimerization. In comparison, residues 42 and 69 reside at the MD-2/TLR4 interaction surface opposite the dimerization interface. Surface charge differences there likely affect the binding angle and/or rigidity between MD-2 and TLR4, exerting an indirect influence on receptor dimerization and activation. Thus, surface charge differences at the two MD-2/TLR4 interfaces determine the species-specific activation of lipid IVA.

Immunology
Inflammation
Innate Immunity
Lipopolysaccharide (LPS)
NF-κB Transcription Factor
Toll-like receptors (TLR)
MD-2
Species Specificity
Lipid IVA

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*

This work was supported, in whole or in part, by National Institutes of Health Grants GM54060 (to D. T. G.) and U19 AI084048 (to J. M. and D. T. G.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and additional references.