The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans;however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. The molecular basis for the recognition of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK), we have expressed mg amounts of the extracellular domain (ECD) of TLR3, and have determined its structure by X-ray crystallography. Double-stranded (ds) RNA, a molecular signature of many viruses, binds and activates TLR3. The structure of TLR3-ECD consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large beta-sheet on the concave surface. The molecule is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan. In solution, purified TLR3-ECD binds dsRNA specifically via a defined ligand-binding site, with an affinity that increases with buffer acidification and ligand size. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers are stabilized by cooperative interactions between the two TLR3-ECDs in a pair, and multiple TLR3-ECD dimers bind to long dsRNAs. The smallest oligonucleotides that form stable complexes with TLR3-ECD (40-50 bp) are also the smallest dsRNAs that activate TLR3 in cells. To determine the molecular basis for ligand binding and signaling, we isolated, crystallized, and solved the structure of the TLR3 signaling complex, consisting of two TLR3-ECD molecules bound to one 46 bp dsRNA oligonucleotide. The glycan-free surfaces of two TLR3-ECDs in the complex face one another on opposite sides of the dsRNA molecule which lies between them. The overall structure of mTLR3-ECD:dsRNA complex contains a two-fold symmetry axis, dictated by the inherent two fold symmetry of the ligand. No conformational change was observed in either the receptor or the ligand upon ligand binding, suggesting that dimerization per se is the activation signal for TLR3. Three intermolecular contacts on the glycan free surfaces of the two TLR3-ECDs stabilize the complex, two protein-dsRNA interactions, and one homotypic interaction between the two TLR3-ECD molecules. The two dsRNA binding sites are widely separated on the glycan free surface of the TLR3-ECD horseshoe, one near the N-terminal end, and one closer to the C-terminus. The two C-terminal sites in the complex are directly across from each other while the N-terminal sites are outstretched at opposing ends of the linear dsRNA molecule. The length of the complex is 141 , which corresponds well with the minimal dsRNA oligo size required for complex formation. The two TLR3-ECD molecules of the complex also bind each other at the C-terminal capping domain, which accounts for dimer formation. The relative importance of the amino acid residues to binding have been determined by mutation analysis. Point mutations in any one of the three interaction sites abrogate dsRNA binding to TLR3 protein and TLR3 activation by dsRNA in cells, indicating a mechanism in which three low affinity sites act cooperatively to form a stable signaling complex.
| Jelinek, Ivett; Leonard, Joshua N; Price, Graeme E et al. (2011) TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection. J Immunol 186:2422-9 |
| Botos, Istvan; Segal, David M; Davies, David R (2011) The structural biology of Toll-like receptors. Structure 19:447-59 |
| Wang, Yan; Liu, Lin; Davies, David R et al. (2010) Dimerization of Toll-like receptor 3 (TLR3) is required for ligand binding. J Biol Chem 285:36836-41 |
| Leonard, Joshua N; Bell, Jessica K; Segal, David M (2009) Predicting Toll-like receptor structures and characterizing ligand binding. Methods Mol Biol 517:55-67 |
| Botos, Istvan; Liu, Lin; Wang, Yan et al. (2009) The toll-like receptor 3:dsRNA signaling complex. Biochim Biophys Acta 1789:667-74 |
| Liu, Lin; Botos, Istvan; Wang, Yan et al. (2008) Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320:379-81 |
| Leonard, Joshua N; Ghirlando, Rodolfo; Askins, Janine et al. (2008) The TLR3 signaling complex forms by cooperative receptor dimerization. Proc Natl Acad Sci U S A 105:258-63 |
