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.

In contrast to T cell receptors, signal transducing cell surface membrane molecules involved in the regulation of responses by cells of the innate immune system employ structures that are encoded in the genome rather than generated by somatic recombination and that recognize either classical MHC-I molecules or their structural relatives (such as MICA, RAE-1, or H-60). Considerable progress has recently been made in our understanding of molecular recognition by such molecules based on the determination of their three-dimensional structure, either in isolation or in complex with their MHC-I ligands. Those best studied are the receptors that are expressed on natural killer (NK) cells, but others are found on populations of T cells and other hematopoietic cells. These molecules fall into two major structural classes, those of the immunoglobulin superfamily (KIRs and LIRs) and of the C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize, in a functional context, the structures of the murine and human molecules that have recently been determined, with emphasis on how they bind different regions of their MHC-I ligands, and how this allows the discrimination of tumor or virus-infected cells from normal cells of the host.