title: N-terminal binding site in the human toll-like receptor 3 ectodomain.
abstract: 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 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 TLR3 ECD revealed that TLR3 activation depends on a single binding site on the nonglycosylated surface in the C-terminal region that includes H539 and several asparagines in LRRs 17 to 20. The localization of TLR3 within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for the ligand binding of TLR3. To clarify the pH-dependent binding mechanism of TLR3 at the structural level, we focused on some highly conserved histidine residues clustered at the N-terminal region of the TLR3 ECD. Mutagenesis approach showed that these residues were essential for the ligand-dependent activation of TLR3 in a cell-based assay. Furthermore, the binding of dsRNA to recombinant TLR3 ECD was strongly pH-dependent, and the binding was reduced by these mutations, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism and that a second dsRNA binding site is present in the N-terminal region in the characteristic solenoid 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.
authors.affiliation: Department of Material and Biochemical Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Watanabe
authors.forename: Takashi
authors.initials: T
authors.lastname: Tokisue
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Tsujita
authors.forename: Misako
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Seya
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nishikawa
authors.forename: Tsunemi
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Kotaro
authors.initials: K
authors.lastname: Fukuda
mesh_terms: D001665:Binding Sites; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009154:Mutation; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D051196:Toll-Like Receptor 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D012330:RNA, Double-Stranded; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D006639:Histidine