TLR4 Ectodomain Research Articles

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.

TLR4 polymorphisms and disease susceptibility

Toll-like receptors (TLRs) play a central role in the regulation of the host immune system. Each TLR recognizes specific pathogen-associated molecular patterns (PAMPs). TLR4 is one of the well characterized pathogen recognition receptors (PRRs) that recognizes the lipopolysaccharide (LPS) of Gram-negative bacteria, some conserved structures from fungal to mycobacterial pathogens and some endogenous ligands. A complex signaling cascade initiates after the ligand binds to the TLR4 ectodomain, leading to the activation of multiple inflammatory genes. Genetic variations greatly influence immune responses towards pathogenic challenges and disease outcome. In this review, we summarize various reports regarding TLR4 polymorphisms and disease susceptibility.

Calcineurin subunit B upregulates β‐interferon production by phosphorylation of interferon regulatory factor 3 via Toll‐like receptor 4

Toll-like receptors (TLRs) function as pattern-reorganization receptors in mammals and play an essential role in innate immunity. After recognizing certain ligands, TLRs activate a cascade of signal pathways to establish a guard environment. For the first time, we report that in vitro treatment with recombinant calcineurin subunit B (CNB) upregulated several TLR-related genes. Calcineurin subunit B interacted with the ectodomain of TLR4 in vitro. On further investigation, phosphorylation of interferon regulatory factor 3 and degradation of IκB-α were observed by CNB stimulation. In addition to pro-inflammatory cytokines, transcription and production of β-interferon were also enhanced after CNB stimulation. Thus, CNB could be further explored as a cancer and virus immunotherapy drug.

Endotoxin·albumin complexes transfer endotoxin monomers to MD-2 resulting in activation of TLR4

Response to Gram-negative bacteria (GNB) is partially mediated by the recognition of GNB-derived endotoxin by host cells. Potent host response to endotoxin depends on the sequential interaction of endotoxin with lipopolysaccharide binding protein (LBP), CD14, MD-2 and TLR4. While CD14 facilitates the efficient transfer of endotoxin monomers to MD-2 and MD-2·TLR4, activation of MD-2·TLR4 can occur in the absence of CD14 through an unknown mechanism. Here, we show that incubation of purified endotoxin (E) aggregates (Eagg, M r ≥ 20 million) in PBS with ≥ 0.1% albumin in the absence of divalent cations Ca2+ and Mg2+, yields E·albumin complexes ( M r ∼70,000). E·albumin transfers E monomers to sMD-2 or sMD-2·TLR4 ectodomain (TLR4ecd) with a ‘ Kd’ of ∼4 nM and induces MD-2·TLR4-dependent, CD14-independent cell activation with a potency only 10-fold less than that of monomeric E·CD14 complexes. Our findings demonstrate, for the first time, a mechanistic basis for delivery of endotoxin monomers to MD-2 and for activation of TLR4 that is independent of CD14.

Association of Inherited Variation in Toll-Like Receptor Genes with Malignant Melanoma Susceptibility and Survival

The family of Toll-like receptors (TLRs) is critical in linking innate and acquired immunity. Polymorphisms in the genes encoding TLRs have been associated with autoimmune diseases and cancer. We investigated the genetic variation of TLR genes and its potential impact on melanoma susceptibility and patient survival. The study included 763 cutaneous melanoma cases recruited in Germany and 736 matched controls that were genotyped for 47 single nucleotide polymorphisms (SNPs) in 8 TLR genes. The relationship between genotype, disease status and survival was investigated taking into account patient and tumor characteristics, and melanoma treatment. Analysis of 7 SNPs in TLR2, 7 SNPs in TLR3 and 8 SNPs in TLR4 showed statistically significant differences in distribution of inferred haplotypes between cases and controls. No individual polymorphism was associated with disease susceptibility except for the observed tendency for TLR2-rs3804099 (odds ratio OR = 1.15, 95% CI 0.99–1.34, p = 0.07) and TLR4-rs2149356 (OR = 0.85, 95% CI 0.73–1.00, p = 0.06). Both polymorphisms were part of the haplotypes associated with risk modulation. An improved overall survival (Hazard ratio HR 0.53, 95% CI 0.32–0.88) and survival following metastasis (HR 0.55, 95% CI 0.34–0.91) were observed in carriers of the variant allele (D299G) of TLR4-rs4986790. In addition various TLR2, TLR4 and TLR5 haplotypes were associated with increased overall survival. Our results point to a novel association between TLR gene variants and haplotypes with melanoma survival. Our data suggest a role for the D299G polymorphism in the TLR4 gene in overall survival and a potential link with systemic treatment at stage IV of the disease. The polymorphic amino acid residue, located in the ectodomain of TLR4, can have functional consequences.

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.

Molecular cloning and structural characterization of the porcine TLR4 ectodomain.

Toll-like receptor 4 (TLR4) regulates innate and adaptive immune responses in pigs. However, the porcine TLR4 gene and structure have not been well-characterized. The ectodomain of porcine TLR4 (pTLR4-ECD) was cloned from a pool of 15 pigs in Taiwan and analyzed. Based on genetic and protein analyses, 5 nucleotide substitutions (A961G, A963G, A1027C, T1606G, A1824G) and three amino acid substitutions (H321 R, K343Q, F535L) were noted. This is the first report documenting the appearance of the hot spot substitutions H321R and K343Q, which are found in the leucine-rich repeat (LRR) of pTLR4. According to three-dimensional modeling, the pTLR4-ECD structure is altered by these amino acid substitutions. These mutations may disturb the binding affinity between pTLR4 and its structural partner MD2. An understanding of the correlation between pTLR4 polymorphisms and immune responses will be important for selecting breeds and developing vaccines in the pork industry.

Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation. In addition, siRNA to Unc93b1 reduced the secretion of the TLR3 ectodomain (T3ECD) into the cell medium. Furthermore, two human single nucleotide polymorphisms that affected herpesvirus and influenza virus encephalopathy as well as a natural isoform generated by alternative splicing were found to be impaired for T3ECD secretion and decreased the abundance of TLR3 on the cell surface. The locations of the SNP P554S and the deletion in the isoform led to the identification of a loop in the TLR3 ectodomain that is required for secretion and a second whose presence decreased secretion. Finally, a truncated protein containing the N-terminal 10 leucine-rich repeats of T3ECD was sufficient for secretion in an Unc93b1-dependent manner.

Recognition of Nucleic Acids by Toll-Like Receptors and Development of Immunomodulatory Drugs

Microbial as well as endogenous nucleic acids are recognized by a group of endosomal Toll-like receptors TLR3, TLR7, TLR8 and TLR9. Recent discoveries significantly improved our understanding of molecular mechanism of their activation and their physiological role. Those include recognition of dsRNA through two nucleic acid binding sites of TLR3 ectodomain, activation of TLR9 by phosphodiester backbone of ssDNA, independent of the nucleotide sequence and phosphorothioate modified bonds, and the role of proteolysis in activation of TLR9. In addition, proteins that chaperone nucleic acids, such as HMGB1 or LL-37, have been described to mediate TLR activation. There is growing evidence that supports involvement of endosomal TLRs in a number of autoimmune diseases, suggesting a therapeutic potential of immunomodulatory endosomal TLR ligands. So far, inhibitory nucleic acids against TLR7, TLR8 and TLR9 as well as small compounds targeting downstream signal transduction of single or several endosomal TLRs have been reported. TLR-targeting drugs have been included in clinical trials as vaccine adjuvants or as therapeutic agents for the treatment of diseases, ranging from cancer, infections, asthma and allergy to autoimmune diseases.

Expression of Functional D299G.T399I Polymorphic Variant of TLR4 Depends More on Coexpression of MD-2 Than Does Wild-Type TLR4

Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.T399I TLR4, we used endotoxinCD14 and endotoxinMD-2 complexes of high specific radioactivity to measure: 1) interaction of recombinant MD-2TLR4 with endotoxinCD14 and TLR4 with endotoxinMD-2; 2) expression of functional MD-2TLR4 and TLR4; and 3) MD-2TLR4 and TLR4-dependent cellular endotoxin responsiveness. Both wild-type and D299G.T399I TLR4(ecd) demonstrated high affinity (K(d) approximately 200 pM) interaction of endotoxinCD14 with MD-2TLR4(ecd) and endotoxinMD-2 with TLR4(ecd). However, levels of functional TLR4 were reduced up to 2-fold when D299G.T399I TLR4 was coexpressed with MD-2 and >10-fold when expressed without MD-2, paralleling differences in cellular endotoxin responsiveness. The dramatic effect of the D299G.T399I haplotype on expression of functional TLR4 without MD-2 suggests that cells expressing TLR4 without MD-2 are most affected by these polymorphisms.

Toll-Like Receptor 4 Decoy, TOY, Attenuates Gram-Negative Bacterial Sepsis

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using ‘the Hybrid leucine-rich repeats (LRR) technique’. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-κB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins.

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.

Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle

Toll like receptors (TLRs) are a class of pattern recognition receptors belonging to the innate immune system. Mutations in the protein coding region of TLRs are associated with altered responsiveness to pathogen-associated molecular patterns (PAMPs). A search was performed for novel mutations in bovine TLR1, TLR2 and TLR4 genes associated with the Mycobacterium avium subsp. paratuberculosis (MAP) infection. The work was also focused on the assessment of linkage between well known mutations in TLR genes (TLR2: Arg677Trp, Pro681His and Arg753Gln; TLR4: Asp299Gly and Thr399Ile), and the susceptibility of cattle to MAP infection. Detection of MAP infection in cattle population ( n = 711) was based on IS900 PCR, which revealed 22.50% ( n = 160) MAP positivity. Known mutations in TLR2 and TLR4 genes were not found in cattle population. A novel mutation Val220Met was associated (Odd's ratio, OR-3.459) with increased susceptibility to MAP infection. Toll/interleukin-1 receptor (TIR) domain of TLR2 was screened for the presence of mutations, wherein a novel Ile680Val mutation was linked with MAP infection. In silico analysis of the bovine TLR4 ectodomain (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. LRR11 of the TLR4 showed five missense mutations possibly linked with the increased susceptibility to MAP infection. The most critical position that may alter the pathogen recognition of TLR molecule was 4th residue downstream to LRR domain. Two such missense mutations in TLR4 (Asp299Asn downstream to LRR11, and Gly389Ser downstream to LRR15) were associated with MAP infection. Briefly, the work describes novel mutations in the bovine TLRs and presents their association with the MAP infection.

Significance of the N-terminal Histidine-rich Region for the Function of the Human Toll-like Receptor 3 Ectodomain

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments such as endosomes and signals in response to virus-derived double-stranded RNA (dsRNA). TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To clarify the pH-dependent binding mechanism of TLR3 at the structural level, we focused on 3 highly conserved histidine residues clustered at the N-terminal region of the TLR3 ectodomain (ECD): H39, H60 and H108. Mutagenesis of these residues showed that H39, H60, and H108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to the recombinant TLR3 ECD depended strongly on pH and dsRNA length, and was reduced by mutations of H39, H60, and H108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA that incorporates this second binding site.

Isolation of Monomeric and Dimeric Secreted MD-2: ENDOTOXIN·sCD14 AND TOLL-LIKE RECEPTOR 4 ECTODOMAIN SELECTIVELY REACT WITH THE MONOMERIC FORM OF SECRETED MD-2

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 E.MD-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 E.sCD14 to form monomeric E.MD-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 E.MD-2. These findings demonstrate directly that the monomeric form of sMD-2 is the active species both for reaction with E.CD14 and TLR4, as needed for potent endotoxin-induced TLR4 activation.

Modulation of Double-stranded RNA Recognition by the N-terminal Histidine-rich Region of the Human Toll-like Receptor 3

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments, such as endosomes, and initiates signals in response to virus-derived double-stranded RNA (dsRNA). The TLR3 ectodomain (ECD), which is implicated in dsRNA recognition, is a horseshoe-shaped solenoid composed of 23 leucine-rich repeats (LRRs). Recent mutagenesis studies on the TLR3 ECD revealed that TLR3 activation depends on a single binding site on the nonglycosylated surface in the C-terminal region, comprising H539 and several asparagines within LRR17 to -20. TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To elucidate the pH-dependent binding mechanism of TLR3 at the structural level, we focused on three highly conserved histidine residues clustered at the N-terminal region of the TLR3 ECD: His39 in the N-cap region, His60 in LRR1, and His108 in LRR3. Mutagenesis of these residues showed that His39, His60, and His108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to recombinant TLR3 ECD depended strongly on pH and dsRNA length and was reduced by mutation of His39, His60, and His108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD characteristic solenoid. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA, which incorporates this second binding site.

Receptor-dsRNA-Receptor

Toll-like receptors recognize molecules associated with pathogens and initiate inflammatory responses. For example, Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), an intermediate viral replication. The TLR3 ectodomain binds as a dimer to dsRNA, but the molecular basis for signaling remains unclear. Liu et al . now report the structure of a complex between two mouse TLR3 ectodomains and dsRNA. Two horseshoe-shaped TLR3-ectodomain monomers bind to opposite faces of the dsRNA through their N and C termini and dimerize through their C termini so that the N termini are at opposite ends of the linear dsRNA molecule. This dimerization mode could mediate signal transduction by facilitating dimerization of the receptor cytoplasmic domains. L. Liu, I. Botos, Y. Wang, J. N. Leonard, J. Shiloach, D. M. Segal, D. R. Davies, Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science 320 , 379-381 (2008). [Abstract] [Full Text]

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.

The TLR3 signaling complex forms by cooperative receptor dimerization

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

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.