Crystal Structures of Human MD-2 and Its Complex with Antiendotoxic Lipid IVa

Ohto, Umeharu; Fukase, Koichi; Miyake, Kensuke; Satow, Yoshinori

doi:10.1126/science.1139111

Cited by 430 publications

(472 citation statements)

References 30 publications

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“…TLR4 could play a secondary role in ligand binding because residues in MD-2 (C95 and C105), which are important for TLR4 binding (11,14), are located at the rim of the ligand-binding cavity (8). This is supported by the higher LPS affinity of the TLR4-MD-2 complex compared with MD-2 on its own (15).…”

supporting

confidence: 56%

“…Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7,8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13).…”

mentioning

confidence: 57%

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Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

114

137

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LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure ( L ipopolysaccharide molecules are complex glycolipids that form the outer layer of the outer membrane of Gramnegative bacteria (1). The lipid A domain of LPS is responsible for cellular activation and consists of a disaccharide to which various substituents, including acyl chains of variable length and number, are attached (2). Escherichia coli lipid A is usually hexa-acylated whereas a tetra-acylated lipid A, lipid IVa, is also produced by E. coli as an intermediate in the lipid A biosynthetic pathway (2). Lipid IVa was originally identified as an inhibitor at the human LPS receptor and was considered a candidate to be developed for clinical use as an endotoxin antagonist. Lipid A signals to host cells through a transmembrane complex consisting of the lipid-binding protein MD-2 and the type 1 receptor TLR4 (1, 3-5). MD-2 is probably the key player in lipid A recognition whereas TLR4, unlike other TLRs, is thought not to participate directly in lipid A binding (5).LPS and lipid A are believed to be recognized by MD-2 following transfer from CD14, which does not participate in the signaling complex (6). Contrary to expectations, ligand binding does not significantly alter the overall structure of MD-2 (7, 8), but the ligands used in the crystallographic studies (lipid IVa and eritoran) are antagonists to human MD-2/TLR4, so it remains unclear what happens to MD-2 and TLR4 upon agonist binding and activation. Active ligands such a lipid A (9) presumably induce structural rearrangements that trigger dimerization of TLR4 and initiate signal transduction (7, 10 -13). Mutagenesis studies identified amino acids 79 -83, 121-124, 125-129 (12), K128, and K132 of human MD-2 as being important in lipid A binding (13), but in the crystal structure of the inactive MD-2-lipid IVa complex, only residues I46, L78, I80, and F121-I124 contact the ligand. The other residues...

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supporting

confidence: 56%

mentioning

confidence: 57%

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Walsh

Gangloff

Monie

et al. 2008

The Journal of Immunology

114

137

View full text Add to dashboard Cite

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“…In the crystal structure of the TLR4-MD-2 complex, the four saturated acyl chains of Eritoran fully occupy the MD-2 hydrophobic pocket, suggesting a prominent role of C12-C14 acyl chains in the binding mechanism [16]. Crystal structures of MD-2 and its complex with the tetraacylated lipid A core of LPS have been determined at 2-resolution [17]. The four saturated C12-C14 acyl chains of the ligand fully occupy MD-2a large hydrophobic cavity.…”

Section: Stimulation Of DC By Dic14-amidine Liposomes Depends On Tlr4mentioning

confidence: 99%

DiC14‐amidine cationic liposomes stimulate myeloid dendritic cells through Toll‐like receptor 4

et al. 2008

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DiC14-amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14-amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14-amidine liposomes induced the production of IL-12p40 and TNF-a by mouse bone marrowderived DC. DiC14-amidine liposomes also activated human DC, as shown by synthesis of IL-12p40 and TNF-a, accumulation of IL-6, IFN-b and CXCL10 mRNA, and up-regulation of membrane expression of CD80 and CD86. DC stimulation by diC14-amidine liposomes was associated with activation of NF-jB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14-amidine liposomes use Toll-like receptor 4 to elicit both MyD88-dependent and Toll/IL-1R-containing adaptor inducing interferon IFN-b (TRIF)-dependent responses.

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“…specific glycolipids) bind. The recently reported x-ray structure of MD-2 has confirmed the presence in MD-2 of a ␤-barrel immunoglobin-fold structure (17). In contrast to the very stable and potently bioactive properties of the monomeric E⅐MD-2 complex (4, 18), recombinant MD-2 expressed and secreted from mammalian cell cultures, such as human embryonic kidney (HEK) 293 cells, is recovered mainly as inactive multimers with little monomer detected (19 -22).…”

mentioning

confidence: 55%

“…The remarkable stability of E⅐MD-2 as a monomeric complex and its water solubility suggest that occupation of the hydrophobic cavity of MD-2 by the acyl chains of endotoxin can significantly increase the stability and solubility of monomeric MD-2. Other hydrophobic compounds, including certain free fatty acids, can occupy the hydrophobic cavity of MD-2 (17) and, at high enough concentrations, may have similar stabilizing effects on sMD-2 monomer. The Express Five TM medium contains several detergents, surfactants, and other lipids that could be responsible for the greater recovery of sMD-2 monomer following size exclusion chromatography (Fig.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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Potent cell activation by endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). MD-2 plays an essential role by bridging endotoxin (E) recognition initiated by lipopolysaccharide-binding protein and CD14 to TLR4 activation by presenting endotoxin as a monomeric E⅐MD-2 complex that directly and potently activates TLR4. Secreted MD-2 (sMD-2) exists as a mixture of monomers and multimers. Published data suggest that only MD-2 monomer can interact with endotoxin and TLR4 and support cell activation, but the apparent instability of MD-2 has thwarted efforts to more fully separate and characterize the individual species of sMD-2. We have taken advantage of the much greater stability of sMD-2 in insect culture medium to fully separate sMD-2 monomer from dimer by gel sieving chromatography. At low nanomolar concentrations, the sMD-2 monomer, but not dimer, reacted with a monomeric complex of E⅐sCD14 to form monomeric E⅐MD-2 and activate HEK293/ TLR4 cells. The monomer, but not dimer, also reacted with the ectodomain of TLR4 with an affinity comparable with the picomolar affinity of E⅐MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E⅐CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.Invasion by Gram-negative bacteria (GNB) 3 is specifically detected and responded to by mammals through mobilization of the innate immune system. In many strains and species of GNB, this process depends on host recognition of and response to the unique complex glycolipid, endotoxin (lipooligosaccharide (LOS) or lipopolysaccharide (LPS)), that comprises much of the outer leaflet of the outer membrane of GNB (1, 2). Minute amounts of endotoxin (E), presented either as an integral part of the outer membrane of GNB or as large aggregates of extracted and purified endotoxin, can stimulate pro-inflammatory responses (2-5). This sensitivity depends upon an ordered series of endotoxin-protein and protein-protein interactions that include the host proteins LPS-binding protein (LBP), membrane-bound or soluble (s)CD14, secreted or TLR4-associated MD-2, and TLR4 (6 -8). Engagement of endotoxin-rich membranes or isolated endotoxin aggregates by LBP facilitates the extraction of endotoxin monomers by CD14 to form monomeric E⅐CD14 complexes that are the most efficient substrate for transfer of endotoxin to MD-2 (4, 6, 9). The monomeric E⅐MD-2 complex is necessary and sufficient to induce TLR4-dependent cell activation by endotoxin (4, 6, 10). MD-2 associates noncovalently with the N-terminal ectodomain of TLR4 and plays a pivotal role not only in TLR4 activation but also in trafficking of TLR4 to the cell surface (7, 11-13). MD-2 also likely interacts transiently with CD14 facilitating transfer of endotoxin monomer from CD14 to MD-2 and, at high molar excess of CD14, reverse transfer of endotoxin from MD-2 to CD14 (14). Simultaneous engagement by MD-2...

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Crystal Structures of Human MD-2 and Its Complex with Antiendotoxic Lipid IVa

Cited by 430 publications

References 30 publications

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

DiC14‐amidine cationic liposomes stimulate myeloid dendritic cells through Toll‐like receptor 4

Isolation of Monomeric and Dimeric Secreted MD-2

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