The dsRNA binding site of human Toll-like receptor 3

Bell, Jessica K.; Askins, Janine; Hall, Pamela R.; Davies, David R.; Segal, David M.

doi:10.1073/pnas.0603245103

Cited by 205 publications

(159 citation statements)

References 28 publications

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“…1A). H539E and N541A were inactive in signaling assays (11,14), and as shown in Fig. 7, these mutants were unable to bind dsRNA at all pH values tested.…”

mentioning

confidence: 87%

“…TLR3 Stimulation-TLR3 activation was followed using an NF-B reporter system as described (11,15). Briefly, HEK293 cells (2.5 ϫ 10 4 in 100 l/well) were plated in 96-well plates and cultured overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Vector Construction and Site-directed Mutagenesis-Mutations of human TLR3 in pUNO (InvivoGen) were made using a QuikChange site-directed mutagenesis kit (Stratagene) as described (10,11). DNA encoding monomeric YFP (a gift from Dr. Susan Pierce, NIAID) with an N-terminal GGGGGG linker was inserted into BamHI and NheI sites at the 3Ј-end of each TLR3 construct.…”

Section: Methodsmentioning

confidence: 99%

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Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

Wang

Liu

Davies

et al. 2010

Journal of Biological Chemistry

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TLR3 (Toll-like receptor 3) recognizes dsRNA, a potent indicator of viral infection. The extracellular domain of TLR3 dimerizes when it binds dsRNA, and the crystal structure of the dimeric complex reveals three sites of interaction on each extracellular domain, two that bind dsRNA and one that is responsible for dimer formation. The goal of this study was to determine which amino acid residues are essential for forming a stable receptor⅐ligand complex and whether dimerization of TLR3 is required for dsRNA binding. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs, we identified the essential interacting residues and determined that the simultaneous interaction of all three sites is required for ligand binding. In addition, we show that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. We conclude that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity receptor⅐ligand complex.Many viruses produce dsRNA at some stage in their replication cycle. Although short stretches of dsRNA are normally found in the microRNA, tRNA, and rRNA of most cells, only viruses synthesize long dsRNA molecules. Therefore, dsRNA longer than ϳ30 bp can serve as a potent molecular signature of viral infection in higher organisms, including man. Consequently, dsRNA is recognized by a number of pattern recognition receptors of the innate immune system, including TLR3 (Toll-like receptor 3) (1-3). TLR3 is a type I transmembrane receptor with an N-terminal extracellular domain (ECD), 2 a single transmembrane helix, and a C-terminal cytoplasmic signaling domain of the TIR (Toll/IL-1 receptor) family. The ECD of TLR3 is located in the interior of endosomes, where it encounters and binds dsRNA and transduces signals that initiate inflammatory and adaptive antiviral responses. dsRNA enters the endosomes either by direct uptake from the medium or by phagocytosis of virally infected cells (4), and as the dsRNA transits from early to late endosomes, the pH decreases progressively from pH ϳ6.2 to 5.5 (5).Recent crystallographic studies have shown how TLR3 recognizes dsRNA at the molecular level (for a review, see Ref. 6). The TLR3 ECD can be described as a coil comprising 23 tandem leucine-rich repeat (LRR) motifs bent into the shape of a horseshoe and capped at the N-and C-terminal ends by specialized structures known as the LRR-NT and LRR-CT domains, respectively (7,8). The TLR3 ECD is decorated with 15 N-linked glycans and has one surface that is devoid of glycan and free to interact with dsRNA. Recombinant TLR3 ECD protein binds dsRNA under mildly acidic conditions, at pH values similar to those found in early and late endosomes (9). Although the TLR3 ECD is monomeric in solution, it binds as dimers to 45-bp segments of dsRNA, and several dimers can bind to long dsRNA strands (9). The x-ray structure of ...

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“…1A). H539E and N541A were inactive in signaling assays (11,14), and as shown in Fig. 7, these mutants were unable to bind dsRNA at all pH values tested.…”

mentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

Wang

Liu

Davies

et al. 2010

Journal of Biological Chemistry

Self Cite

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“…Others and we have shown that the central LRRs of these receptors are involved in agonist recognition and have proposed that an interaction between these receptors on the concave side of the solenoid would be sterically improbable. Indeed, a recent model based on the mutagenesis work of TLR3 bound to dsRNA proposes an interaction of this agonist with the convex side of receptor dimers (25). Thus, TLRs may interact with cognate agonists in a different manner from that of other LRR proteins such as the RNase inhibitor protein, the Nogo receptor, and the glycoprotein 1b receptor that bind their cognate ligands on the concave side of the molecule (26 -28).…”

Section: Discussionmentioning

confidence: 99%

The Polymorphism P315L of Human Toll-Like Receptor 1 Impairs Innate Immune Sensing of Microbial Cell Wall Components

Omueti

Mazur²,

Thompson³

et al. 2007

The Journal of Immunology

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As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9–12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.

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“…Despite the finding of potential dsRNA binding surfaces through the examination of the TLR3 ectodomain crystal structure (45,46), evidence of direct binding is lacking. It is possible that other proteins serve to bridge dsRNA interactions with TLR3, as well as mda-5.…”

Section: I:c) Protects Mice From Lethal Ptv Infection In 3-to 4-wk-olmentioning

confidence: 99%

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

Gowen

Wong

Jung

et al. 2007

The Journal of Immunology

102

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In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C12U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C12U) therapy and failed to produce IFN-α, IFN-β, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3−/− mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C12U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

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The dsRNA binding site of human Toll-like receptor 3

Cited by 205 publications

References 28 publications

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

The Polymorphism P315L of Human Toll-Like Receptor 1 Impairs Innate Immune Sensing of Microbial Cell Wall Components

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

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