Responsiveness to different types of endotoxins by combinations of MD-2 and TLR4.

<p>Responsiveness to different types of endotoxins by combinations of MD-2 and TLR4.</p

Mutations of murine MD-2 residues E122 and L125 diminish responsiveness to tetraacylated endotoxin while preserving activation by hexaacylated endotoxin.

<p>(A) Murine MD-2 mutants mE122K and mE122K L125K gain the ability to activate the human TLR4 with hexaacylated endotoxin. (B) Murine MD-2 mutants mE122K and mE122K L125K in combination with murine TLR4 exhibit reduced activation by tetraacylated lipid IVa.</p

Large amino acid residue side chains at position 82 in hMD-2 augment hydrophobic interactions and promote TLR4 activation by hypoacylated endotoxins.

<p>(A) The surface representation of human MD-2 (2E56, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107520#pone.0107520-Ohto1" target="_blank">[28]</a>) and mutants at position 82. (left) wt hMD-2, (middle) hMD-2_V82M, (right) hMD-2_V82F. The surface at the amino acid residue at position 82 of wt MD-2 and both mutants is colored in grey, yellow or green, respectively. Green translucent area on the wt MD-2 (left) represents the difference in surface area occupied in the hMD-2_V82F mutant. (B) The structure of human MD-2 (2E59 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107520#pone.0107520-Ohto1" target="_blank">[28]</a>) with bound lipid IVa. At position 82 side chains and the corresponding molecular surfaces are shown for the wt hMD-2 valine (blue) and phenylalanine mutant (green). Introduction of a large phenylalanine 82 side chain causes clash with a buried acyl chain of lipid IVa (right, close-up view). Figures were prepared with the USCF Chimera package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107520#pone.0107520-Pettersen1" target="_blank">[26]</a>.</p

Species-Specific Activation of TLR4 by Hypoacylated Endotoxins Governed by Residues 82 and 122 of MD-2

<div><p>The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.</p></div

The structure of the human MD-2 with bound endotoxin.

<p>(A) Human MD-2 (pdb id 3FXI, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107520#pone.0107520-Park1" target="_blank">[27]</a>) is shown in grey ribbon. The side chains of amino acid residues at positions 82 (valine), 122 (lysine) and 125 (lysine) are shown in stick representation. These amino acid residues are positioned at the entrance to the binding pocket and come in close proximity to the ligand (here the hexaacylated endotoxin, shown in yellow). Figure was prepared with the USCF Chimera package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107520#pone.0107520-Pettersen1" target="_blank">[26]</a>. (B) Amino acid alignment of residues at positions 82, 122 and 125 of human, murine and equine MD-2 (red – acidic amino acid; blue – basic amino acid; green – nonpolar/hydrophobic amino acid). (C) The structure of lipid A. The arrows indicate acyl chains that are absent in the structure of the msbB endotoxin and in lipid IVa.</p

Human MD-2 mutants hV82F and hK122R respond to tetraacylated endotoxin.

<p>(A) Human TLR4 does not support activation by tetraacylated endotoxin regardless of MD-2 species or mutations. (B) Specific mutations of human MD-2 enable responsiveness to tetraacylated endotoxin in combination with murine TLR4. (C) Human MD-2 mutants hV82F and hK122R can support IL-8 production by mTLR4-expressing HEK293 cells in response to lipid IVa stimulation. #p>0,1; *p<0,1; **p<0,01 (t-test, compared to mock-stimulated control). (D, E) Mutations of hMD-2 at residues 122 and 125 strongly affect the ability to mediate endotoxin activation. #p>0,1 (not significant); *p<0,1; **p<0,01; ****p<0,0001 (t-test, hMD-2 mutants or mMD-2 stimulated with 1000 ng/ml lipid IVa compared to wt hMD-2 stimulated with 1000 ng/ml lipid IVa).</p

Activation of human TLR4 by metal ions requires different TLR4 and MD-2 residues than activation by endotoxin.

<p>(A) hTLR4 amino acid residues that are essential for activation by LPS are not needed for activation by nickel or cobalt. HEK293 cells were transfected with luciferase reporter plasmids, plasmid encoding hMD-2 and plasmid encoding either wild type hTLR4 or the hTLR4 mutant hF440A. (B) hMD-2 amino acid residues that are essential for activation by LPS are not needed for activation by nickel or cobalt. HEK293/hTLR4 cells were transfected with luciferase reporter plasmids and with plasmid encoding either wild type or mutant hMD-2. #p≥0,01 (not significant); *p<0,01; **p<0,001; (t-test, compared to the unstimulated control (as indicated by brackets) or compared to the wt hTLR4 with the corresponding treatment).</p

Nickel and cobalt activate HEK293 cells via human TLR4 and require MD-2 for activation.

<p>(A) HEK293/hTLR4 cells were transfected with the luciferase reporter plasmids with or without the plasmid encoding hMD-2. After stimulation, the luciferase activity was measured. (B) Mouse TLR4 with either mouse or human MD-2 does not support activation by nickel or cobalt. HEK293/mTLR4 cells were transfected with the luciferase reporter plasmids with or without plasmid encoding MD-2. (C) Mutation of histidines at positions 456 and 458 in hTLR4 nearly abolishes responsiveness to nickel and cobalt ions. HEK293 cells were transfected with luciferase reporter plasmids, plasmid encoding hMD-2 and plasmid encoding either wild type or mutant hTLR4. Luciferase activity was measured as indicated in Methods. #p≥0,01 (not significant); *p<0,01; **p<0,001; ***p<0,0001 (t-test, compared to the unstimulated control). The chart legend applies to all three panels.</p

Nickel and cobalt activate MyD88-dependent and -independent pathways.

<p>HEK293/hTLR4 cells were transfected with luciferase reporter plasmids (constitutive Renilla-luciferase reporter and inducible) (A) IFNβ-responsive or (B) IP-10-responsive firefly-luciferase reporter) and with plasmid encoding wild type hMD-2. *p<0,01; **p<0,001; (t-test, compared to the unstimulated control). The chart legend applies to both panels.</p

MD-2 provides crucial stabilization that supports the formation of TLR4/MD-2 heterodimer with nickel or cobalt ions.

<p>(A) A ribbon representation of a TLR4/MD-2 heterodimer (pdb id 3fxi). Amino acid residues of both TLR4 and MD-2 that form hydrophobic interactions with each other are shown as spheres and indicated with arrows. (B) A ribbon representation of the intrinsic dimerization interface between both TLR4 ectodomains. Amino acid residues that engage in direct interactions with one another are shown as spheres. Histidines H431, H456 and H458 are represented as sticks and colored orange. Green spheres represent metal ions that are coordinated between the indicated histidines. (C) A ribbon representation of TLR4 dimer without MD-2 shown from a top view. Without MD-2 both TLR4 ectodomains lack proper stabilization and can wobble around one another, causing disruption of a proper conformation that would enable cytoplasmic TIR domain dimerization and consecutive triggering of the TLR4 receptor signaling pathway. Figures were prepared with the UCSF Chimera package [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120583#pone.0120583.ref031" target="_blank">31</a>].</p