Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation. Lysine ubiquitination, catalysed by E3 ubiquitin ligases, is pivotal for regulating protein stability and cell signalling. Using protein semisynthesis, the roles of the C-terminal carboxylate and conformational interconversion in HECT-domain E3 catalysis are now characterized, revealing evolutionary plasticity in side chain versus backbone utilization.