Structural and biochemical analysis of cullin-based ubiquitin ligases reveal regulatory mechanisms of ubiquitination machinery

Ubiquitin-dependent proteolysis controls protein stability and plays an important regulatory role in a broad spectrum of biological processes. At the core of the ubiquitination process are the ubiquitin ligases (UBL) that function as the central component of the ubiquitination pathway by not only catalyzing the final transfer of ubiquitin from an ubiquitin conjugating enzyme to the substrate, but also governing the specificity of the modification reaction. One class of UBLs is the multi-subunit ubiquitin ligase Skp1-Fbox-Cul1 -Roc1 (SCF). The exact mechanism of assembly this complex has not been well understood until recently when Cand1 was identified. Cand1 is a 120-kDa HEAT repeat protein that forms a tight complex with the Cul1-Roc1 SCF catalytic core, inhibiting the assembly of the multi-subunit ligase. The mechanism by which this is done was unknown. Therefore, I solved the crystal structure of the Cand1-Cul1-Roc1 complex, which shows Cand1 adopting a curved superhelical structure, clamping around the elongated SCF scaffold protein Cul1. This structure showed that Cand1 inserts a beta-hairpin into an adaptor-binding site on Cul1, inhibiting its interactions with the Skp1protein. Simultaneously Cand1 buries a lysine residue on Cul1, whose modification by the ubiquitin-like protein, Nedd8, is able to block Cand1-Cul1 association. Together with biochemical evidence, these structural results elucidate the mechanisms by which Cand1 and Nedd8 regulate the assembly-disassembly cycles of SCF and other cullin-based E3 complexes. In the presence of Cand1 Cul1 cannot be neddylated. However, I determined that neddylation could occur if Skp1 and an F-box protein are present. Together these structural and biochemical details added greater detail to our understanding of SCF regulation.; Finally, I performed biochemical analysis on Cul3 based ubiquitination machinery. The Kelch-like ECH-associated protein 1 (Keap1) BTB-domain containing protein binds to Cul3 and to its substrate (Nrf2) simultaneously, serving comparative roles to Skp1-Fbox proteins concurrently. A fully functional Nrf2 ubiquitination system was achieved using all recombinant proteins. The Cul3 system was also used to identify pathways for human Dishevelled3 ubiquitination.