Structural basis of toll-like receptor 3 signaling with double-stranded RNA

Abstract

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

Fig 1

dsRNA:TLR3 signaling complex. Mouse TLR3 ectodomains (green and cyan) form a dimer on the dsRNA (blue and red). The N glycans are shown (light green and light blue). (A) The N- and C-terminal binding sites. (B) Illustration of how the two C-terminal domains are brought together in the complex. Figures generated with PyMol (DeLano Scientific, San Carlos, CA)

Fig 2

The dsRNA binding sites of TLR3. (A) Residues involved in the interaction on the C-terminal site. (B) Illustrates the NF-κB activity of human TLR3 mutants stimulated with pI:pC. (C) Residues involved in binding at the N-terminal site. Note that each interaction involves two strands of the RNA. The dsRNA molecule may undergo a screw rotation of plus or minus one base pair in the crystal.

Fig 3

Closeup of the C-terminal domain interacting residues. Some of these residues (678–681) are located on a conserved loop observed in other TLR structures.

Fig 4

Model of the full-length dsRNA:TLR3 signaling complex. The proximity of the two C-termini permits association of the trans-membrane helices and the dimerization of the cytoplasmic TIR domains. The TIR domains were homology modeled from the structure of the TLR10 TIR domains (pdb code 2J67).