There is an urgent need to understand the mechanisms by which protein ubiquitination controls eukaryotic biology and human diseases and to identify novel therapeutic strategies. The regulation of protein ubiquitination is largely achieved by SCF ubiquitin E3 ligases (E3s) that bind substrates through different, interchangeable F-box subunits, thereby controlling substrate abundance and/or activity. A remarkable feature of many F-box proteins is that they often have a modular structure and that each protein can recruit a specific subset of target proteins to an SCF E3 for ubiquitination. For example, the F-box protein Fbx4 recognizes the proto-oncoprotein cyclin D1 and the telomeric DNA-binding protein TRF1, while Fbxo31 mediates cyclin D1 degradation after DNA damage. The broad, long-term objective of this research plan is to understand how individual F-box subunits can recognize and bind substrate proteins that vary in amino-acid sequence and tertiary structure, and how multiple F-box proteins can act on a single target. Our overarching hypothesis is that the target specificity and diversity of SCF E3s are determined by the structural and interaction properties of F-box subunits with their targets. In this project we aim to delineat the structural mechanisms and molecular logic underlying the selectivity of Fbx4 and Fbxo31 towards their respective substrates cyclin D1 and TRF1. This knowledge will facilitate efforts to design new agents that interact with SCF E3s in specific and therapeutically beneficial manners. The Specific Aims are: 1. To determine the molecular mechanism of the Fbx4-TRF1 interaction and regulation of Fbx4 dimerization. We will a) characterize in detail the structural properties of the Skp1-Fbx4-TRF1 complex and identify specific amino-acid side chains involved in the E3-substrate interaction; b) generate a series of rationally designed Fbx4 mutants and evaluate their effects on the Fbx4-TRF1 interaction, Fbx4 dimerization and TRF1 degradation; and c) determine correlations between the structural and functional effects of the mutations. 2. To determine the interaction and partnering mechanism of Fbx4 with B-crystallin and cyclin D1. We will a) determine the crystal structure of the Skp1-Fbx4-B-crystallin-cyclin D1 phosphopeptide complex and define the structural principles underlying the Fbx4 substrate selectivity for cyclin D1 over TRF1 using NMR and other biophysical methods; b) perform structure-activity studies of the Fbx4/B-crystallin/cyclin D1 interactions and determine the role of B-crystallin in cyclin D1 ubiquitination and its regulation using a fully reconstituted in vitr cyclin D1 ubiquitination system and cell-based assays. 3. To elucidate the structural properties of Fbxo31 both free and bound to cyclin D1. We will a) characterize the detailed structural properties of Fbxo31 and identify the determinants of the Fbxo31-cyclin D1 interaction; b) determine the role of interacting interfacial residues in the Fbxo31-cyclin D1 complex in the binding and degradation of cyclin D1; and c) determine the extent to which the Fbxo31/Fbx4-cyclin D1 interaction targets specific lysine residue(s) for ubiquitination and thus regulates the proteasomal degradation of cyclin D1.