The TLR3 signaling complex forms by cooperative receptor dimerization

Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):258-63. doi: 10.1073/pnas.0710779105. Epub 2008 Jan 2.

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Binding Sites
  • Buffers
  • Cell Line
  • Cell Separation
  • Chromatography, Gel
  • Cross-Linking Reagents / pharmacology
  • DNA, Single-Stranded / chemistry
  • Dimerization
  • Flow Cytometry
  • Humans
  • Hydrogen-Ion Concentration
  • Ligands
  • Models, Biological
  • Oligonucleotides / chemistry
  • RNA, Double-Stranded / chemistry*
  • Toll-Like Receptor 3 / chemistry
  • Toll-Like Receptor 3 / metabolism*
  • Toll-Like Receptor 4 / chemistry

Substances

  • Buffers
  • Cross-Linking Reagents
  • DNA, Single-Stranded
  • Ligands
  • Oligonucleotides
  • RNA, Double-Stranded
  • TLR3 protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 3
  • Toll-Like Receptor 4