Toll-like receptors (TLRs) and receptors for pro-inflammatory cytokines IL-1 and IL-18 share a common TIR domain in their intracellular region and belong to the TLR/IL1-R superfamily. TLRs recognize pathogen-associated molecular patterns (PAMPs) to initiate protective immune responses. The molecular pathways for these receptors are complex and their dysregulation is associated with many human diseases both within and beyond the immune system. Signal transduction of these receptors is initiated by the approximation of the receptor TIR domains upon binding of PAMPs and cytokines. This leads to the recruitment of intracellular TIRcontaining adaptors such as MyD88, TIRAP/Mal, TRIF and TRAM. MyD88 is critical for signaling responses of IL-1, IL-18, and all TLRs except TLR3. In addition to its C-terminal TIR domain, MyD88 contains an N-terminal death domain (DD). Through the DD, MyD88 interacts with IRAKs, including IRAK1, IRAK2, IRAK4 and IRAK-M, which are characterized by an Nterminal DD and a C-terminal Ser/Thr kinase or kinase-like domain. Eventually, the ensuing pathway activates transcription factors NF-κB, AP-1, and IRFs to elicit anti-pathogen responses and inflammation. Despite the biological importance of the TLR/IL-1R signaling system, limited structural and mechanistic information is available. In this application for a MERIT extension, we propose to continue our studies in this system using structural methods such as X-ray crystallography and cryo-electron microscopy, as well as pharmacological and light microscopy approaches. In the extension period, we will focus on four related areas: 1. Elucidating the molecular mechanism of TIR-TIR interactions in wild-type and disease mutant adaptor proteins. We will build from our preliminary data in this regard and pursue high-resolution structure determination of these oligomeric complexes. 2. Interrogating TLR/IL-1R signal transduction using reconstituted systems on lipid bilayers and total internal reflection fluorescence microscopy. These studies will better mimic cellular signaling because they provide the membrane-based, two-dimensional platform for signal complex assembly. In our preliminary data, we have established the feasibility of reconstitution and imaging. 3. Elucidating TLR/IL-1R signal transduction and the functional interaction with other innate immune pathways in CRISPR-modified macrophages in which an endogenous signaling protein is replaced by the fluorescently tagged version. We have already generated a number of these cell lines and shown that they can be activated. We will image these cells under different activating conditions to obtain quantitative analysis on the temporal and spatial control of these receptors. 4. Identifying small molecule inhibitors for IRAKs in the treatment of lymphomas. We have previously crystallized IRAK4 and revealed an inactive conformation of the kinase during the process of trans-autophosphorylation. In our preliminary data, we have crystallized...