Structural Model of MD-2 and Functional Role of Its Basic Amino Acid Clusters Involved in Cellular Lipopolysaccharide Recognition

Gruber, Anton; Manček, Mateja; Wagner, Hermann; Kirschning, Carsten J.; Jerala, Roman

doi:10.1074/jbc.m400993200

Cited by 117 publications

(118 citation statements)

References 48 publications

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“…Der p2, p1, f 2), GM2-activator protein, Npc2, and their orthologs (16). The recent publication of the crystal structure of recombinant MD-2 (17) confirms earlier structural models of MD-2 that were based on the solved structures of other ML domain proteins (31)(32)(33)(34)(35)(36)(37). A hallmark of MD-2, as well as other ML domain proteins, is the presence of a deep, hydrophobic cavity that can expand to accommodate specific (glyco)lipid ligands.…”

Section: Discussionsupporting

confidence: 59%

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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Section: Discussionsupporting

confidence: 59%

Isolation of Monomeric and Dimeric Secreted MD-2

Teghanemt

Widstrom

Gioannini

et al. 2008

Journal of Biological Chemistry

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“…MD-2 is associated with the extracellular domain of TLR4 (22,24) and has been shown to directly interact with LPS (25). The LPS binding site of MD-2 is comprised of several clusters of basic residues and a hydrophobic region, which presumably exist as a pocket (26,27). Thus, MD-2 can establish complementary interactions with the negatively charged phosphate groups and hydrophobic acyl chains of the lipid A portion of LPS.…”

Section: Discussionmentioning

confidence: 99%

Prothymosin-α inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction

Mosoian

Teixeira

Burns

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8 + T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8 + cells. Thus, a protein produced by CD8 + T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTα may provide therapeutic leads for IFN-sensitive viruses.

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“…Preparation of Recombinant MD-2 WT and MD-2C133F-Recombinant MD-2 was produced in Escherichia coli as described previously (8), using solubilization of inclusion bodies followed by their purification and refolding on reversed phase column chromatography. Biological activity of MD-2 was tested on HEK293 cells transfected with TLR4.…”

Section: Methodsmentioning

confidence: 99%

“…MD-2 has been indispensable in almost all investigated conditions of TLR4-triggered inflammation; therefore, it could represent the "Achilles' heel" of the inflammatory response to LPS and a target for a pharmacological intervention in endotoxemia as well as other conditions involving cell activation mediated by TLR4 (4,5). The existence of a single free cysteine residue among the seven cysteine residues has been predicted from MD-2 mutagenesis (6, 7) and molecular modeling proposed that Cys 133 lies in the hydrophobic pocket (8,9). The hydrophobic binding site of MD-2 was also mapped by an apolar probe, bis-ANS, which does not contain acyl chains as most LPS antagonists yet preserves the characteristic structural motif of lipid A, consisting of a hydrophobic region and a pair of separated negatively charged groups (10).…”

mentioning

confidence: 99%

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

Manček-Keber

Gradišar

Pestaña

et al. 2009

Journal of Biological Chemistry

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MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys 133 and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor ␣ production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation. TLR4 is a receptor for lipopolysaccharide (LPS)4 a major constituent of outer membrane of Gram-negative bacteria. MD-2 is the final LPS-binding protein in the recognition cascade before TLR4 transmits the signal across the cell membrane to activate the inflammatory response. MD-2 binds to the ectodomain of TLR4 and binds LPS either alone or in complex with TLR4 (1, 2). Mice deficient in MD-2 survive endotoxic shock (3). MD-2 has been indispensable in almost all investigated conditions of TLR4-triggered inflammation; therefore, it could represent the "Achilles' heel" of the inflammatory response to LPS and a target for a pharmacological intervention in endotoxemia as well as other conditions involving cell activation mediated by TLR4 (4, 5). The existence of a single free cysteine residue among the seven cysteine residues has been predicted from MD-2 mutagenesis (6, 7) and molecular modeling proposed that Cys 133 lies in the hydrophobic pocket (8, 9). The hydrophobic binding site of MD-2 was also mapped by an apolar probe, bis-ANS, which does not contain acyl chains as most LPS antagonists yet preserves the characteristic structural motif of lipid A, consisting of a hydrophobic region and a pair of separated negatively charged groups (10). Crystal structures of MD-2 with bound eritoran or lipid IVa confirmed the location of Cys 133 in the hydrophobic pocket in close vicinity of bound lipid A derivatives (11, 12). The free cysteine residue inside the binding pocket can thus be a target for irreversible inhibition of MD-2 activity. An inhibitory mechanism based on a covalent modification of a free cysteine residue in the active or binding site of a protein has been demonstrated for other proteins, s...

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Structural Model of MD-2 and Functional Role of Its Basic Amino Acid Clusters Involved in Cellular Lipopolysaccharide Recognition

Cited by 117 publications

References 48 publications

Isolation of Monomeric and Dimeric Secreted MD-2

Isolation of Monomeric and Dimeric Secreted MD-2

Prothymosin-α inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction

Free Thiol Group of MD-2 as the Target for Inhibition of the Lipopolysaccharide-induced Cell Activation

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