Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran

Kim, Ho Min; Park, Beom Seok; Kim, Jung-In; Kim, Sung Eun; Lee, Judong; Oh, Se Cheol; Enkhbayar, Purevjav; Matsushima, Norio; Lee, Hayyoung; Yoo, Ook Joon; Lee, Jie-Oh

doi:10.1016/j.cell.2007.08.002

Cited by 990 publications

(1,120 citation statements)

References 56 publications

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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].…”

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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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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“…Despite the overwhelming data on the TLR4-mediated signaling cascade (29) as well as the solved crystal structure of the ectodomain of TLR4 in complex with MD-2 and its agonistic and antagonistic ligands, LPS and lipid IVa, respectively (15,16), the mechanisms governing receptor activation and its dynamics remain unclear. Structural information is available for the soluble domains.…”

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confidence: 99%

“…The ectodomain of TLR4 is composed of 22 repeats of a leucine-rich repeat module, adopting a solenoidal conformation (15,16). A single transmembrane helix connects the ectodomain to the Toll/ IL-1R (TIR) cytoplasmic domain, responsible for intracellular signal propagation upon receptor activation by LPS (2,17).…”

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confidence: 99%

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

Panter

Jerala

2011

Journal of Biological Chemistry

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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.Pattern recognition receptors are proteins expressed by cells of the innate immune system and act as a first line of host defense against invading microorganisms, recognizing diverse but highly conserved structural pathogen-associated molecular patterns of bacteria, fungi, and viruses (1). Activation of pattern recognition receptors by their respective ligands triggers an immediate immune response, characterized by proinflammatory cytokine production (1, 2). Toll-like receptors (TLRs) 2 are a family of pattern recognition receptors central to the vertebrate innate immune response. Activation of TLRs links innate and adaptive immunity through proinflammatory signaling and induction of costimulatory molecules on immune cells (3, 4). However, activation of TLRs has to be a tightly regulated process. TLRs have to be able to mount an immediate immune response upon binding of the agonist, whereas in the absence of the ligand it is imperative that they remain in an inactive state to prevent unwanted activation that may lead to excessive inflammation and autoimmune disease (5). This is especially important for TLR4, the cellular receptor for LPS, a molecular signature of the outer cell membrane of Gram-negative bacteria (6). Activation of TLR4 by LPS affects the regulation of more than 1000 genes (7). Additionally, TLR4 signaling has been implicated in sterile inflammation through activation of the receptor by endogenous ligands (8 -14).TLR4 is a type I transmembrane glycoprotein. The ectodoma...

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“…For example, lipopeptides are detected by the leucine-rich repeat (LRR) 4 region 9 -12 of TLR1/6 (16,17), and residues D295 and D367 of TLR5 are important for the recognition of bacterial flagellin (16). MD-2 binds to TLR4 on a lateral surface of the TLR4 solenoid near to the N terminus (18). Interestingly, TLR4 single nucleotide polymorphisms (D299G and T399I) in humans are located far away from the N-terminal MD-2 binding site but reduce LPS responsiveness (19,20).…”

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confidence: 99%

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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Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran

Cited by 990 publications

References 56 publications

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

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

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

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

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