Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.
Toll-like receptors (TLRs) detect pathogens and endogenous danger, initiating immune responses that lead to the production of proinflammatory cytokines. At the same time, TLR-mediated inflammation is associated with a number of pathological states, including infectious, autoimmune, inflammatory, cardiovascular and cancer-related disorders. This dual role of the pathways has attracted widespread interest from pharmaceutical industries. Cytoplasmic signalling by TLRs starts by their TIR (Toll/interleukin-1 receptor) domain interacting with TIR domain-containing adaptor proteins MyD88, MAL, TRIF and TRAM. Combinatorial recruitment of these adaptors via TIR:TIR domain interactions orchestrates downstream signalling pathways, leading to induction of the proinflammatory genes. Although many constituents of the TLR pathways have been identified, the available information on their coordinated interactions is limited. Such information is crucial for a mechanistic understanding of TLR signalling, development of therapeutic strategies, and understanding of the molecular basis of the consequences for human disease of adaptor polymorphic variants. We have discovered that TIR domains can form large assemblies. We hypothesized that TIR domain signalling occurs through a mechanism involving higher-order assembly formation. In this study we aim to determine the molecular architecture of higher-order assemblies formed by TIR domains with a focus on TRAM-TRIF assemblies in the TLR4 and TLR3 pathway.
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