The dsRNA binding site of human Toll-like receptor 3

Abstract

Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.

Fig. 1.

Two sulfate ions bind to TLR3-ECD. (A) View of the concave and glycan-free lateral surfaces of TLR3-ECD (Protein Data Bank ID code 2A0Z). The molecular surface is shown in gray, N-linked glycan is in yellow, and two bound sulfate ions are in red. (B and C) Detailed views of residues coordinating the sulfate ions with hydrogen bonds indicated by dotted lines. (B) At site 1, the sulfate ion is coordinated by N361, Y326, and H359. (C) At site 2, the sulfate is coordinated directly by R488, N515, Q538, and H539 and by a water-mediated hydrogen bond (W52) to E570.

Fig. 2.

Mutation of His-539 to Glu in sulfate site 2 abrogates TLR3 signaling. (A) Mutation of residues coordinating sulfate 1 (site 1) and sulfate 2 (site 2). HEK293 cells, transfected with WT or mutant TLR3, were stimulated or not with p(I):p(C) and assayed for NF-κB activation. Results (±SEM) are reported as fold induction relative to unstimulated cells. Only H539E was significantly different from WT (∗, P < 0.05). (B) HEK293 cells were cotransfected with 40, 4, or 0.4 ng of TLR3 DNA and stimulated (filled bars) or not (open bars) with p(I):p(C). NF-κB activation measurements, reported as relative luciferase units (±SEM), are averaged from at least three separate experiments. Note that transfection with 40 ng of DNA gives constitutive activation with all plasmids except for the empty vector control.

Fig. 3.

Mutation of residues near H539 further define the dsRNA binding site. (A and B) Residues mutated on the concave and lateral surfaces proximal to H539 are highlighted in green and blue, respectively. The two residues important for function, H539 and N541, are colored magenta. The boxed area in A is shown in detail in B. (C) Transfected cells were examined for responsiveness to pI:pC as in Fig. 2A. Bars are color-coded as in A. Only N541A showed significant loss of responsiveness (∗, P < 0.05).

Fig. 4.

H539E and N541A fail to bind ligand. TLR3-ECD (WT or mutant) protein was incubated with p(I):p(C) or buffer and analyzed by size-exclusion chromatography. Column fractions were assayed for TLR3-ECD by ELISA at 450 nm. Data are representative of two separate experiments. Elution profiles of WT (Top), H539E (Middle), and N541A (Bottom) TLR3-ECD protein are shown. Open bars, protein alone; filled bars, protein plus p(I):p(C). Arrows indicate the elution volumes of pI:pC and TLR3-ECD when run alone.

Fig. 5.

Role of LRR insertions and basic residue patches in dsRNA recognition. (A) Location of mutated residues. Groups of positively charged residues in patches 1 and 2, shown in blue, were mutated to alanine. The inserts in LRRs 12 and 20, shown in green, were deleted. (B) Responsiveness of TLR3 mutants to pI:pC. Deletions of LRR inserts are shown in green, and mutants in the positively charged patches are color-coded blue. In patch 1, mutant Δ4 contains four mutations, R65A, K89A, K117A, and K147A; Δ6 contains six mutations, R65A, K89A, K117A, K137A, K139A, and K147A; and Δ8 contains eight mutations, R65A, K89A, K117A, K137A, K139A, K145A, K147A, and K165A. In patch 2, Δ3 contains three mutations, K335A, K345A, K371A; and Δ7 contains seven mutations, R331A, K335A, K345A, K371A, R394A, K416A, and K418A. ∗, significant loss in activity (P < 0.05).

Fig. 6.

Model of TLR3 recognition of dsRNA and signaling complex. (A) Diagram of TLR3 ligand recognition and signaling complex. dsRNA is depicted in gray, and TLR3-ECD is shown in blue. H539 and N541 are shown as magenta and green circles, respectively. (B) A 19-bp dsRNA molecule (gray surface, Protein Data Bank ID code 1QC0) was docked onto the TLR3-ECD structure (blue). In the model, H539 (magenta) coordinates a phosphate group in the minor groove whereas N541 (green) forms a hydrogen bond to the 2′OH of the ribose also in the minor groove. The LRR20 insertion is shown in cyan. Two TLR3-ECD molecules, related by an ≈180° rotation, bind to one dsRNA ligand. Multiple TLR3 molecules could bind to a longer dsRNA as illustrated in A. (C) A proposed signaling complex of full-length TLR3 includes the oligomer model of ligand interaction from B rotated 45° with 22-residue transmembrane helices and TIR domains (Protein Data Bank ID code 1FYV). Single receptors are shown in blue or orange. Linker regions between the LRR-CT and TM (7 residues) and the TM and TIR domain (19 residues) are denoted by black dotted lines. The homologous position of the Lps^d mutation in the BB loop of the TLR4 TIR domain (25), postulated to interact with MyD88 (26), is indicated in red. Notably, the separation of the TLR3-ECD in the proposed oligomer easily accommodates the formation of a TIR dimer (27).