Project Summary Post-translational modification of proteins by ubiquitin (Ub) is a regulatory mechanism that controls nearly all aspects eukaryotic cell biology. Ubiquitination alters properties of target proteins such as stability, subcellular localization, intermolecular interactions, and activity and thereby regulates processes such as cell cycle control, DNA repair, signal transduction, and immunity. The relevance of Ub signaling to human health is underscored by the fact that its dysregulation is implicated in pathologies such as cancer, cardiovascular disease, and immune disorders and that it is a validated target for therapeutic intervention in cancer with FDA-approved medications extending the lives of multiple myeloma patients. Ub signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3, which act in tandem to conjugate Ub to target proteins. The largest family of E3s, with more than 200 members, are cullin-RING E3 ligases (CRLs) which are modular, multi-subunit complexes involved in a diverse array of biological processes by functioning with the E2 Cdc34 to recognize substrates and catalyze the extension of lysine 48-linked polyubiquitin (K48-polyUb) chains that signal for proteasomal destruction. One of the best characterized CRL subfamilies are Skp, Cullin, F-box (SCF) RING E3 ligases which control cell cycle progression by catalyzing K48-polyUb chains on key regulatory proteins at defined timepoints. CRLs are also of intense interest therapeutically as they are hijacked by proteolysis targeting chimeras (PROTACs) to catalyze K48-polyUb assembly on therapeutically important target proteins of interest. Despite their fundamental importance biologically and therapeutically, many key questions regarding how Cdc34 and SCF/CRL E3s function together at a molecular level remain, including: 1) How do Cdc34/SCF complexes specifically catalyze K48-polyUb as opposed to other types of polyUb chains?, 2) What is the architecture of the complete Cdc34/SCF E3/substrate/Ub donor/Ub acceptor complex during catalysis?, 3) What is the structural basis by which CRLs catalyze PROTAC-mediated ubiquitination of substrate proteins?. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to determine the structural basis by which Cdc34 and SCF function together to specifically assemble Lys48- linked polyUb chains to control the cell cycle (Aim 1) and to determine the molecular mechanisms by which PROTAC molecules with therapeutic potential hijack CRL E3 ligases to target proteins of interest for ubiquitination and proteasomal destruction (Aim 2). Studying the system through this lens provides a unique opportunity to gain deeper insights into both basic mechanisms of Cdc34/SCF/CRL E3 function as well as into the mechanisms and molecular rules governing design of effective PROTACs that could ultimately provide a platform for the development of novel therapeutics for cancer and othe...