This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background We rely on our innate immune system as the first line of defense response against invading pathogens. This immune response is critically dependent on the Toll-like receptors (TLRs). Each TLR recognizes a different molecular pattern that is characteristic of specific pathogens, such as bacterial cell wall components, bacterial filaments, or viral DNA and RNA. Upon ligand binding, TLRs transmit a signal to the nucleus that leads to the production of proinflammatory compounds. These include antimicrobial cytokines, and compounds that recruit the adaptive immune system, which establishes long-term immunity to specific pathogens. Objectives We will determine three-dimensional structures of human TLR5, TLR8 or TLR9, in complex with their ligands[unreadable][unreadable]" bacterial flagellin, single-stranded RNA or CpG DNA, respectively. The structures of these complexes will provide critical insight into the molecular basis of pathogen recognition and proinflammatory signal generation. We will express the ectodomains of TLR5/8/9 in milligram quantities and crystallize them in complex with their respective ligands. We will determine three-dimensional structures of the complexes by X-ray crystallography. To test our structure-based hypotheses on TLR function, we will measure the effect on immune signaling of engineered mutations that are predicted from the structures to interfere with ligand binding or signal generation. We propose a strategy to seek high-affinity TLR ligands, or agonists. Relevance Our work will reveal the molecular basis for how pathogen recognition is translated into an immune response signal. Our structures will guide efforts to design synthetic TLR agonists, which could serve as novel vaccine adjuvants, or as immunomodulatory therapeutics. Such therapeutics would provide a powerful new means to prevent and treat infectious diseases.