Interactions Within the E2 Enzyme Cdc34-Ubiquitin Complex Are Transient

Abstract

The process of ubiquitylation involves the transfer of the small 76-residue protein ubiquitin (Ub) between a series of enzymes (E1, E2, E3) until it binds to a lysine residue of the substrate protein. When repeated several times, the E1-E2-E3 cascade forms a K48-linked polyubiquitin (poly-Ub) chain targeting the protein substrate for 26S proteasomal degradation. The E2 enzyme is the key protein in this cascade as it must recognize both the E1 and E3 enzymes, label the substrate, as well as form a covalent thiolester with Ub. Cdc34 is a class II E2 conjugating enzyme in the Ub-dependent degradation pathway of cell cycle proteins. It is comprised of a ∼170 residue catalytic domain, common to all E2 enzymes, which contains the active site cysteine (C93). Two unique features of cdc34 are its acidic loop (residues 102-113) and acidic tail (residues 171-236), both of which are required to produce poly-Ub chains. However, the underlying mechanism that these regions have on chain assembly remains unclear. We have used NMR spectroscopy to study protein-protein interactions between cdc34 and Ub by using a stable covalent disulphide cdc34-Ub to mimic the thiolester complex. Chemical shift mapping was used to identify sites of non-covalent interactions in the cdc34-Ub to create a model of the complex. Competitive binding studies using free Ub or the cdc34 tail (residues 183-236) with cdc34-Ub indicate that transient non-covalent interactions exist between cdc34 and Ub. These studies provide the first detailed structural information to explain the unique mechanism used by cdc34 to promote poly-Ub chain assembly.