The TLR3 signaling complex forms by cooperative receptor dimerization

*Experimental Immunology Branch, National Cancer Institute,
^†Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, and
^§Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; and
^‡Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Sanger Hall, Room 2-040, 1101 East Marshall Street, Richmond, VA 23298

Contributed by David R. Davies, November 14, 2007 (received for review October 16, 2007)

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.

Footnotes

^¶To whom correspondence may be addressed. E-mail: drd{at}niddk.nih.gov or dave_segal{at}nih.gov
Author contributions: J.N.L., J.K.B., D.R.D., and D.M.S. designed research; J.N.L., R.G., and J.A. performed research; J.A. and D.H.M. contributed new reagents/analytic tools; J.N.L. and R.G. analyzed data; and J.N.L., R.G., and D.M.S. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0710779105/DC1.