E2 enzymes act at the heart of the ubiquitylation cascade, as they interact with both E1 and E3 enzymes. Surprisingly little is known on how the activities of E2 enzymes are regulated. Mounting evidence suggests that E2 enzymes are subject to ligand binding-induced allosteric changes and post-translational modifications that serve regulatory functions. This project aims to reveal the impact of such modifications on the structures, the conformational dynamics, the macromolecular interactions, and, hence, the physiological functions of E2 enzymes. These studies hold the potential of revealing “druggable” protein surfaces/interfaces or allosteric sites that may be exploited for therapeutic purposes.