ABSTRACT Inborn errors of immunity (IEI; or primary immunodeficiencies) comprise a large, heterogeneous group of inherited genetic defects that manifest as acute sensitivity to infection or recurring infection, autoimmunity, autoinflammation, allergies, or malignancy. Even in the case of exceptionally rare IEIs, studies of the impact(s) of these genetic defects can offer an unparalleled opportunity to gain deeper insight into the molecular and cellular mechanisms of fundamental immune system function. Through our established connection to a multi- national clinical monitoring network, we identified an unprecedented IEI arising from a single heterozygous missense mutation in the gene encoding the innate immune protein and double-stranded (ds)RNA sensor oligoadenylate synthetase-like (OASL) which results in a glutamic acid to lysin amino acid substitution at a critical site in the protein structure. This new IEI was identified in a patient with a post-infectious bronchiolitis obliterans triggered by adenovirus (a double-stranded (ds)DNA-virus). This finding and our preliminary investigations implicate OASL gain-of-function and dysregulation of currently uncharacterized connections between innate immune networks sensing dsRNA (via retinoic acid-inducible gene I; RIG-I) and dsDNA (via cyclic GMP-AMP synthase; cGAS) as the basis of this novel IEI. However, the nature of the protein defect and how it leads to aberrant, disease-causing OASL activity is currently unknown. In this exploratory research program, we will test our current working model in which the E237K amino acid substitution changes OASL structure and/ or dynamics in a manner that shifts the balance of OASL innate immune signaling via altered interaction with its binding partners (e.g. dsRNA or cGAS) or altered regulation of their activity (e.g. cGAS activation by dsDNA). The aims of this proposal will address the following two overarching questions: Aim 1– How does the OASL-E273K substitution alter OASL protein structure and dynamics? Aim 2–What is the effect of the OASL-E273K substitution on OASL’s interaction with dsRNA and with cGAS, and on cGAS activation by dsDNA? We will use a powerful combination of approaches to address these questions, including computational, e.g. classical and accelerated molecular dynamics simulations coupled with protein residue pathway analyses, and experimental approaches, e.g. hydrogen-deuterium exchange coupled to mass spectrometry, X-ray crystallographic structure determination, and defined in vitro binding and enzyme activity assays. Collectively, this exploratory project will establish the necessary foundation for detailed future studies that promise to significantly deepen our understanding of OASL’s function(s) as a point of innate immune pathway crosstalk in a cellular context and during infection, as well as the molecular basis of the novel OASL IEI we have identified.