NMR studies of hexaacylated endotoxin bound to wild-type and F126A mutant MD-2 and MD-2·TLR4 ectodomain complexes

Host response to invasion by many gram-negative bacteria depends upon activation of Toll-like receptor 4 (TLR4) by endotoxin presented as a monomer bound to myeloid differentiation factor 2 (MD-2). Metabolic labeling of hexaacylated endotoxin (LOS) from Neisseria meningitidis with [(13)C]acetate allowed the use of NMR to examine structural properties of the fatty acyl chains of LOS present in TLR4-agonistic and -antagonistic binary and ternary complexes with, respectively, wild-type or mutant (F126A) MD-2 ± TLR4 ectodomain. Chemical shift perturbation indicates that Phe(126) affects the environment and/or position of each of the bound fatty acyl chains both in the binary LOS·MD-2 complex and in the ternary LOS·MD-2·TLR4 ectodomain complex. In both wild-type and mutant LOS·MD-2 complexes, one of the six fatty acyl chains of LOS is more susceptible to paramagnetic attenuation, suggesting protrusion of that fatty acyl chain from the hydrophobic pocket of MD-2, independent of association with TLR4. These findings indicate that re-orientation of the aromatic side chain of Phe(126) is induced by binding of hexaacylated E, preceding interaction with TLR4. This re-arrangement of Phe(126) may act as a "hydrophobic switch," driving agonist-dependent contacts needed for TLR4 dimerization and activation.     

Identification of Key Residues That Confer Rhodobacter sphaeroides LPS Activity at Horse TLR4/MD-2

The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.     

Essential Roles of Hydrophobic Residues in Both MD-2 and Toll-like Receptor 4 in Activation by Endotoxin

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4·MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4·MD-2·LPS complex, driving TLR4 activation.Bacterial lipopolysaccharide (LPS)³ is recognized by the innate immune system of vertebrates via an elaborate mechanism involving the membrane receptor TLR4 (1, 2). The extracellular (or cell surface) proteins LPS-binding protein and CD14 promote extraction and transfer of individual molecules of LPS from the Gram-negative bacterial outer membrane to MD-2, either secreted monomeric soluble (s)MD-2 or MD-2 bound with high affinity to the ectodomain of TLR4 (3–7). In contrast to other Toll-like receptors, TLR4 requires an additional molecule, MD-2, for ligand recognition (8). In contrast to MD-2, there has been no evidence of direct binding of LPS to TLR4 (9, 10). Although LPS, and particularly the lipid A portion of LPS, is generally conserved among Gram-negative bacteria, there are many variables in LPS structure that affect TLR4 activation. Most important is the acylation pattern of the lipid A moiety, which represents the minimal segment of LPS that can trigger activation of TLR4 (11). Comparison of crystal structures of MD-2 with and without bound tetraacylated lipid IVa indicates no significant alteration of the protein fold in the absence or presence of bound ligand (12). It has been proposed that both LPS and MD-2 are key to the different effects of tetra- versus hexaacylated LPS on TLR4 (8, 13, 14). Lipid IVa complexed to murine MD-2 has weak agonist effects on murine TLR4 but acts as a receptor antagonist in the same complex containing human MD-2. Hexaacylated endotoxins complexed to human or murine MD-2 act as potent TLR4 agonists. The crystal structure of the TLR4·MD-2·eritoran complex revealed that MD-2 binds to the N-terminal region of TLR4 (15). It seems likely that for TLR4 activation, there needs to be an additional interaction between two ternary TLR4·MD-2·LPS complexes, which is agonist-dependent (15–17). Because tetraacylated and hexaacylated endotoxins that act, respectively, as TLR4 antagonists and agonists differ only in their acylation pattern, we speculated that hydrophobic protein-lipid A interactions are essential in the agonist properties of hexaacylated lipid A. To pursue this hypothesis, we used molecular modeling to select and test the involvement of solvent-exposed hydrophobic residues of MD-2 and TLR4, which we reasoned could be needed for TLR4 activation. We show by mutagenesis studies that residues on the solvent-exposed hairpin of MD-2 support transfer of endotoxin from CD14 to MD-2 and TLR4 activation only when these sites contain hydrophobic residues. In the ectodomain of TLR4, we have identified two neighboring phenylalanine residues located on the convex face of consecutive leucine rich repeats that are required for LPS-triggered TLR4 activation. From those results and molecular docking, we propose that amino acid side chains of both MD-2 and TLR4 ectodomain form an acyl chain binding site, which envelops part of an acyl chain of lipid A that cannot fit into the binding pocket of MD-2 in a TLR4·MD-2 complex and represents a key to LPS-induced TLR4 activation.     

Molecular basis of reduced potency of underacylated endotoxins

Potent TLR4-dependent cell activation by gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.     

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.     

MD-2 Residues Tyrosine 42, Arginine 69, Aspartic Acid 122, and Leucine 125 Provide Species Specificity for Lipid IVA

Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)·MD-2 complex. A synthetic lipid A precursor, lipid IV_A, induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IV_A 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 IV_A species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IV_A. Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IV_A, 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 IV_A. 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 IV_A.     

Novel roles in human MD-2 of phenylalanines 121 and 126 and tyrosine 131 in activation of Toll-like receptor 4 by endotoxin

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E.CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E.CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E.CD14 +/- TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe(121) and Tyr(131) are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E.MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E.CD14 +/- TLR4. E.MD-2(F126A) binds TLR4 with high affinity (K(d) approximately 200 pm) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E.MD-2. These findings reveal roles of Phe(121) and Tyr(131) in TLR4-independent interactions of human MD-2 with E.CD14 and, together with Phe(126), in activation of TLR4 by bound E.MD-2. These findings strongly suggest that the structural properties of E.MD-2, not E alone, determine agonist or antagonist effects on TLR4.     

MD-2 Determinants of Nickel and Cobalt-Mediated Activation of Human TLR4

Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the –independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation.     

N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.     

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

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 Cys¹³³ 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 α 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 Cys¹³³ 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 Cys¹³³ 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, such as in cysteine proteases, where the cysteine residue participates in the catalytic triad (13), in cholesteryl ester transfer protein (CETP) (14), IκB kinase (15), thioredoxin reductase (16), and sortase (17).In our study, we investigated the possibility of targeting free cysteine residue of MD-2 for the inhibition of LPS signaling. We determined covalent binding into the hydrophobic pocket of MD-2 for fluorescent compounds 2-(4′-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and N-pyrene maleimide. Drugs JTT-705 and auranofin, already in use for alternative indications, were also shown to bind to MD-2 and decrease LPS signaling. The identity of Cys¹³³ as the residue responsible for this interaction was demonstrated by point mutagenesis. Our results confirm that the proposed mechanism of inhibition of MD-2 can have potential therapeutic value but may also have a physiological role.     

Functional activity of MD-2 polymorphic variant is significantly different in soluble and TLR4-bound forms: decreased endotoxin binding by G56R MD-2 and its rescue by TLR4 ectodomain

MD-2 is an essential component of endotoxin (LPS) sensing, binding LPS independently and when bound to the ectodomain of the membrane receptor TLR4. Natural variation of proteins involved in the LPS-recognition cascade such as the LPS-binding protein, CD14, and TLR4, as well as proteins involved in intracellular signaling downstream of LPS binding, affect the cellular response to endotoxin and host defense against bacterial infections. We now describe the functional properties of two nonsynonymous coding polymorphisms of MD-2, G56R and P157S, documented in HapMap. As predicted from the MD-2 structure, the P157S mutation had little or no effect on MD-2 function. In contrast, the G56R mutation, located close to the LPS-binding pocket, significantly decreased cellular responsiveness to LPS. Soluble G56R MD-2 showed markedly reduced LPS binding that was to a large degree rescued by TLR4 coexpression or presence of TLR4 ectodomain. Thus, cells that express TLR4 without MD-2 and whose response to LPS depends on ectopically produced MD-2 were most affected by expression of the G56R variant of MD-2. Coexpression of wild-type and G56R MD-2 yielded an intermediate phenotype with responses to LPS diminished to a greater extent than that resulting from expression of the D299G TLR4 polymorphic variant.     

Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa

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 (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.     

Specific high affinity interactions of monomeric endotoxin.protein complexes with Toll-like receptor 4 ectodomain

Potent Toll-like receptor 4 (TLR4) activation by endotoxin has been intensely studied, but the molecular requirements for endotoxin interaction with TLR4 are still incompletely defined. Ligand-receptor interactions involving endotoxin and TLR4 were characterized using monomeric endotoxin.protein complexes of high specific radioactivity. The binding of endotoxin.MD-2 to the TLR4 ectodomain (TLR4ECD) and transfer of endotoxin from CD14 to MD-2/TLR4ECD were demonstrated using HEK293T-conditioned medium containing TLR4ECD+/-MD-2. These interactions are specific, of high affinity (KD<300 pm), and consistent with the molecular requirements for potent cell activation by endotoxin. Both reactions result in the formation of a Mr approximately 190,000 complex composed of endotoxin, MD-2, and TLR4ECD. CD14 facilitates transfer of endotoxin to MD-2 (TLR4) but is not a stable component of the endotoxin.MD-2/TLR4 complex. The ability to assay specific high affinity interactions of monomeric endotoxin.protein complexes with TLR4ECD should allow better definition of the structural requirements for endotoxin-induced TLR4 activation.     

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.     

Crystal structure of CD14 and its implications for lipopolysaccharide signaling

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.     

Partially glycosylated dendrimers block MD-2 and prevent TLR4-MD-2-LPS complex mediated cytokine responses

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.     

Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: combined effects of lipid A acylation state and TLR4 polymorphisms on signaling

The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymorphic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-kappaB-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-kappaB activity in wild-type transfectants under the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-alpha, IL-6, IL-1beta, and IL-10 production. Cytokine levels were significantly lower (approximately 20-90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant.     

MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.     

Transfer of monomeric endotoxin from MD-2 to CD14: characterization and functional consequences

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin depends on sequential transfer of monomers of endotoxin from an aggregated form to CD14 via the lipopolysaccharide-binding protein and then to MD-2. We now show that monomeric endotoxin can be transferred in reverse from MD-2 to CD14 but not to lipopolysaccharide-binding protein. Reverse transfer requires an approximately 1000-fold molar excess of CD14 to endotoxin-MD-2. Transfer of endotoxin from MD-2 to extracellular soluble CD14 reduces activation of cells expressing TLR4 without MD-2. However, transfer of endotoxin from MD-2 to membrane CD14 (mCD14) makes cells expressing MD-2.TLR4 sensitive to activation by the endotoxin-MD-2 complex. An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4. These findings describe a novel pathway of endotoxin transfer that provides an additional layer of regulation of cell activation by endotoxin.