Discovery and characterization of non-canonical ubiquitin conjugating enzymes

Ubiquitin conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation is required to maintain cellular homeostasis and impacts almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a post-translational modification (PTM) that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) Mass Spectrometry (MS), to discover and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We employed structural modelling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results identify the missing E2s required for non-canonical ubiquitylation, highlight the versatility of ubiquitin modifications and challenge the view of ubiquitylation as an exclusively lysine specific PTM.