IL-1 family cytokines are instrumental in orchestrating inflammatory and immune responses to infection. However, dysregulated IL-1 family cytokine signaling is a key contributor to numerous chronic inflammatory diseases and autoimmune disorders. IL-33, an IL-1 family member, is a potent inducer of allergic type 2 immunity. Like other IL-1 family cytokines, it positively impacts human health – it activates a wide range of immune cells in response to microbial invasion, plays important roles in tissue homeostasis and repair, and reverses symptoms in mouse models of Alzheimer’s disease; but also drives negative impacts – it promotes allergic asthma, participates in pathological fibrotic reactions, and is linked to autoimmunity. IL-33 functions by binding to its cognate receptor, ST2, and then recruiting its secondary receptor, IL-1RAcP. The latter receptor is shared by other IL-1 family cytokines, most notably IL-1. We have recently determined the X-ray crystal structure of the murine IL-33/ST2/IL-1RAcP signaling-competent ternary complex. Together with our preliminary mutagenesis, binding and functional analyses, these data suggest the hypothesis that the molecular mechanisms by which IL-33 and IL-1 recruit their shared secondary receptor, IL-1RAcP, differ markedly. This has important implications for the development of therapeutic molecules that can manipulate IL- 33 signaling, either to augment IL-33 activation to promote beneficial physiological effects or to inhibit IL-33 signaling to prevent adverse pathological effects. Our proposed studies are designed to fully demonstrate the differences in molecular mechanisms of IL-1 and IL-33 signaling and to leverage this growing mechanistic knowledge to engineer novel therapeutic activators and inhibitors of IL-33 signaling. In Specific Aim 1, we will determine the structural basis of IL-33 cytokine signaling complex formation. Having determined the crystal structure of the murine IL-33/ST2/IL-1RAcP ternary complex, we will now determine the structure of the human IL-33/ST2/IL-1RAcP ternary complex, which is directly relevant to our planned therapeutic designs. We will also evaluate the solution structures of these complexes by small-angle X-ray scattering (SAXS) and assess their conformational dynamics by hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis and molecular dynamics (MD) simulations. In Specific Aim 2, we will define the molecular basis of shared receptor usage by IL-1 and IL-33. Using a structure-guided approach based on published structures of IL-1/IL-1RI/IL- 1RAcP complexes and our new and forthcoming structures of IL-33/ST2/IL-1RAcP complexes, we will mutate residues within the interfaces formed by the composite cytokine/cognate receptor and accessory protein surfaces, and measure their binding affinities and signaling properties relative to the wild type proteins. In Specific Aim 3, we will develop novel therapeutics by rationally manipulating IL-33 signaling mechanisms....