The dsRNA binding site of human Toll‐like receptor 3

Abstract Toll-like receptor 9 (TLR9) stimulatory CpG-containing oligodeoxynucleotides (ODNs) with phosphorothioate backbones have successfully replaced the naturally occurring agonists of TLR9 in drug development due to their increased stability. Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. Here, we report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. Furthermore, we show that while single point mutations of W47, W96 and K690 within the TLR9 binding site retains full TLR9 activation by phosphodiester-based ODNs, activation by phosphorothioate-based ODNs is strongly impaired. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.

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Microbial Products Stimulate Human Toll-like Receptor 2 Expression through Histone Modification Surrounding a Proximal NF-κB-binding Site

Journal of Biological Chemistry ◽

10.1074/jbc.m705151200 ◽

2007 ◽

Vol 282 (43) ◽

pp. 31197-31205 ◽

Cited By ~ 35

Author(s):

Christopher M. Johnson ◽

Richard I. Tapping

Keyword(s):

Histone Modification ◽

Binding Site ◽

Toll Like Receptor ◽

Toll Like Receptor 2 ◽

Microbial Products

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Duplicated TLR5 of zebrafish functions as a heterodimeric receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719245115 ◽

2018 ◽

Vol 115 (14) ◽

pp. E3221-E3229 ◽

Cited By ~ 9

Author(s):

Carlos G. P. Voogdt ◽

Jaap A. Wagenaar ◽

Jos P. M. van Putten

Keyword(s):

Binding Site ◽

Danio Rerio ◽

Gene Products ◽

Structural Studies ◽

Toll Like Receptor ◽

Complex Structural ◽

Species Specific ◽

Toll Like Receptor 5 ◽

Induced Changes ◽

Receptor Conformation

Toll-like receptor 5 (TLR5) of mammals, birds, and reptiles detects bacterial flagellin and signals as a homodimeric complex. Structural studies using truncated TLR5b of zebrafish confirm the homodimeric TLR5–flagellin interaction. Here we provide evidence that zebrafish (Danio rerio) TLR5 unexpectedly signals as a heterodimer composed of the duplicated gene products drTLR5b and drTLR5a. Flagellin-induced signaling by the zebrafish TLR5 heterodimer increased in the presence of the TLR trafficking chaperone UNC93B1. Targeted exchange of drTLR5b and drTLR5a regions revealed that TLR5 activation needs a heterodimeric configuration of the receptor ectodomain and cytoplasmic domain, consistent with ligand-induced changes in receptor conformation. Structure-guided substitution of the presumed principal flagellin-binding site in human TLR5 with corresponding zebrafish TLR5 residues abrogated human TLR5 activation, indicating a species-specific TLR5–flagellin interaction. Our findings indicate that the duplicated TLR5 of zebrafish underwent subfunctionalization through concerted coevolution to form a unique heterodimeric flagellin receptor that operates fundamentally differently from TLR5 of other species.

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Structural Study of Binding of Flagellin by Toll-Like Receptor 5

Journal of Bacteriology ◽

10.1128/jb.185.14.4243-4247.2003 ◽

2003 ◽

Vol 185 (14) ◽

pp. 4243-4247 ◽

Cited By ~ 50

Author(s):

Saul G. Jacchieri ◽

Ricardo Torquato ◽

Ricardo R. Brentani

Keyword(s):

Binding Site ◽

Electrostatic Interactions ◽

Bacterial Species ◽

Helical Conformation ◽

Theoretical Argument ◽

Toll Like Receptor ◽

Unit Energy ◽

Serovar Typhimurium ◽

Toll Like Receptor 5 ◽

Predicted Binding Site

ABSTRACT In order to predict the binding regions within the complex formed by Toll-like receptor 5 (TLR-5) and flagellin, a complementary hydropathy between the two proteins was sought. A region common to the flagellins of Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Listeria monocytogenes was shown to be hydropathically complementary to the 552-to-561 fragment of TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profile of this region is shared with flagellins of 377 bacterial species out of a total of 723 publicly available sequences. A conformational analysis of the predicted binding site of TLR-5, whose structure is still unknown, was carried out with a methodology already applied to similar problems. To sample the conformations available to the peptide chain, a plot of the number of conformations per unit energy interval (density of states) versus energy was built. Following a theoretical argument, conformations belonging to maxima in this plot were selected. The most stable structure obtained in this search, an α-helical conformation, was shown to form the electrostatic interactions Glu552-Gln89, Asp555-Arg92, and Arg558-Glu93 with the predicted binding site of the flagellin of S. enterica serovar Typhimurium, formed by the 88-to-97 chain fragment (LQRVRELAVQ), which is likewise α helical.

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