The post-translational modification of proteins with ubiquitin has a central role in all eukaryotic cells. As a result, disruption of ubiquitin transfer is associated with diseases including cancer, immunological diseases, and neurological disorders. Proteins can be modified by single ubiquitin moieties or by polyubiquitin chains of eight different linkages, and this spells out a rich code that dictates the fate of the substrate proteins. For example, ubiquitin chains connected by lysine 48 (Lys48) signal for destruction of the substrate by the proteasome, and these chains have a critical role in regulating protein abundance.
The E2 enzyme Ube2k builds only Lys48 chains, and is therefore tightly coupled with degradation of proteins. We have probed how Ube2k can build Lys48 chains by mutating and characterising amino acids around its active site. The results allowed us to model an incoming ubiquitin molecule bound to Ube2k. Using this prediction as a template, we generated protein-based inhibitors of Ube2k. Crystal structures of two Ube2k-inhibitor complexes revealed that the inhibitors both bind at a hydrophobic groove on the E2. Assessment of the mechanism of inhibition suggests they may block activity via multiple mechanisms. These inhibitors are being used as tools for understanding the role of Ube2k in cells.