Linear Clustering of Toll-like receptor 3 on Long dsRNA

Abstract

Toll-like receptors (TLRs) are innate immune receptor that sense pathogen invasion by detecting unique molecular pattern conserved in pathogen and initiates immune response. Among TLR families, TLR3 is localized in endosome, recognizes double strand RNA derived from virus genome or replication intermediates and senses host virus infection. Ligand-induced homodimerization activates TLR3 in acidic conditions. The activated TLR3 recruits adaptor molecules, such as TRIF, initiating an antiviral response by activating transcription factors IRF3, IRF7, and NF-κB, which regulate the expression of cytokines and interferons. Previous biochemical and structural studies revealed that TLR3 requires 40-50 base-pair length of dsRNA for stable dimer formation and activation. However, short dsRNA of this length exhibits only basal activity and fails to initiate TLR3-dependent signaling in in vivo experiments. For robust immune response, exceeding 100 base pair length is required. Additionally, TLR3 exhibits positive cooperativity, where the binding affinity increases with longer dsRNA. To elucidate the ligand length dependency and positive cooperative nature of TLR3-dependent signaling at the molecular level, cryo-electron microscopy (cryo-EM) structures of TLR3 in complex with dsRNA, averaging ~400 base pairs in length, were investigated under various conditions. These structures reveal dimeric TLR3 units linearly aligned along the dsRNA strand with regular direction and spacing. This clustering is mediated by charged residues at the cluster interface region through electrostatic interactions. Perturbing TLR3 cluster formation using blocking antibodies, without affecting ligand binding and dimer formation, significantly reduces TLR3 signaling. This underscores the importance of clustering in TLR3 signaling. In summary, TLR3 forms a linear cluster on dsRNA, providing a signaling platform for the efficient binding of adaptor molecules and the induction of robust signaling.