The TLR3 signaling complex forms by cooperative receptor dimerization

Abstract

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

Fig. 1.

TLR3 binding to dsRNA is saturable, specific, and a function of dsRNA size. Capture (A) and competition (B–D) ELISAs were performed by using immobilized 540-bp dsRNA at pH 6.0. (A) TLR3ecd binds saturably to dsRNA. (B) Soluble polyI:C and dsRNA (139 bp) inhibit the binding of TLR3ecd (1 μg/ml) to immobilized dsRNA at comparable concentrations. (C) dsDNA (532 bp), ssDNA (90 bp), and ssRNA (633 bp) inhibit binding much less effectively than dsRNA (540 bp). (D) Inhibition of TLR3ecd binding to immobilized dsRNA depends upon dsRNA length. Experiments were performed in triplicate, and error bars indicate one standard deviation.

Fig. 2.

Binding affinity is a function of pH and dsRNA size. (A and B) TLR3ecd binding to immobilized dsRNA as determined by SPR. (A) Binding of TLR3ecd (40 μg/ml) to dsRNA (540 bp) increases with acidity. Arrows indicate the points at which TLR3ecd injection began and ended with the change back to buffer alone. (B) Affinity of TLR3ecd (40 μg/ml) binding at pH 6.0 increases with the length of the immobilized dsRNA. (C) The amount of TLR3ecd bound to dsRNA at equilibrium was determined by SPR, at several concentrations of soluble (free) TLR3ecd, and data were plotted for Scatchard analysis. Downward curvature suggests positive cooperativity.

Fig. 3.

Composition of TLR3-dsRNA complexes. (A) The stoichiometry of complexes formed by TLR3ecd and 90-bp dsRNA was measured by titrating TLR3ecd against a fixed dsRNA concentration (1 μM) and analyzing the mixture by gel filtration (GF) at pH 6.0. For this ligand, the stoichiometry is 4:1 (TLR3ecd:dsRNA), because the only peak observed at this ratio corresponds to the complex, and, at higher and lower ratios, unbound TLR3ecd and dsRNA were apparent. (B) The stoichiometries of complexes formed by dsRNA of different lengths as determined by GF. Ligands smaller than 48 bp did not form stable complexes (SI Fig. 6). Sedimentation equilibrium (C) and sedimentation velocity analyses (D) of TLR3ecd and dsRNA (48 bp) mixtures at pH 5.5. At a 2:1 loading ratio, a single species is observed with the molecular mass expected for a complex that consists of two TLR3ecd and one dsRNA molecule (SI Appendix 1). Standard errors are indicated in C. Note that in A and D, peak heights reflect the fact that TLR3ecd's coefficient of absorption is less than that of dsRNA. Mixture compositions in A, C, and D are reported as the molar ratio of TLR3ecd:dsRNA. The predicted molecular mass of 48-bp dsRNA is 31.1 kDa.

Fig. 4.

Receptor cross-linking activates TLR3. (A) Intracellular and surface expression of TLR3 in stable reporter cell lines, as assessed by FACS; shaded histograms are isotype controls. (B) TLR3-mediated activation of NF-κB in reporter cells by dsRNA (139 bp, 5 μg/ml) or polyI:C (1 μg/ml) and inhibition by bafilomycin. (C) TLR3 activation after cross-linking at the cell surface with anti-TLR3 polyclonal antibodies (pAb). (D) Cells preincubated in bafilomycin (baf) were activated by anti-TLR3 pAb (0.1 μg/ml) but not by dsRNA (as in B). Experiments were performed in triplicate, and error bars indicate one standard deviation.

Fig. 5.

Activation of TLR3 depends on dsRNA size and receptor localization. Cells were stimulated with dsRNA ligands of varying length. mo, medium only. Experiments were performed in triplicate, and error bars indicate one standard deviation.