In untreated patients, systemic lupus erythematosus (SLE) can manifest as chronic inflammation, organ damage, and nephritis. Monogenic SLE provides clues to understanding this complicated and heterogeneous disease. Mutations that disrupt the functionality of the endonuclease Dnase1L3 lead to anti-nuclear antibody (ANA) accumulation and high incidence of lupus nephritis. Dnase1L3 has the unique capacity to degrade chromatin DNA and DNA microparticles. Since SLE patients often show reduced Dnase1L3 activity, therapeutic Dnase1L3 has the potential to serve as an SLE therapy. To make Dnase1L3 a cost-effective enzyme replacement therapy, we propose modifying the surface of recombinant human Dnase1L3 with poly(ethylene glycol) PEG. PEGylation is a common technique to extend the serum half-life of therapeutic proteins. To guide our selection of PEG conjugation sites that maintain activity, we solved the X-ray crystal structure of the core enzyme of Dnase1L3 to 1.9Å resolution. While the core enzyme structure highlights the differences between Dnase1L3 and Dnase1, the unique activity of Dnase1L3 is dependent on its C-terminal Domain (CTD). The CTD confers nuclease activity on chromatin and microparticle DNA via an unknown mechanism. To understand the capacity for the CTD of Dnase1L3 to prevent SLE pathogenesis, we have undertaken a combined functional/structural approach. Our central hypothesis is that targeted PEGylation of Dnase1L3 will enhance the pharmacokinetic properties of the enzyme without compromising activity. In addition, the interaction of the CTD with DNA complexes confers unique activity as a DNA recruitment domain. We will test our hypothesis with two specific Aims. Aim 1 will test a set of selected surface residues on Dnase1L3 for sites amenable to PEGylation. Conjugating PEG to surface residues typically improves the pharmacokinetics properties of therapeutically useful proteins. Using our crystal structure as a template, we have defined ten sites for PEG conjugation based on defined biophysical criteria. Aim 2 will determine the structure, function of the CTD, and model the holoenzyme structure. Building on our circular dichroism, SAXS, and NMR data, we will quantitate the potential structural transitions of the CTD of Dnase1L3 that may act to recruit DNA. Due to our Dnase1L3 over-expression and purification system, and the preliminary data from our crystal structure, we are uniquely equipped to enhance the Dnase1L3 enzyme for SLE treatment while establishing the molecular mechanisms of serum DNA clearance that control SLE pathogenesis.