Structural and biophysical characterisation of ubiquitin variants that specifically inhibit the ubiquitin conjugating enzyme Ube2d2

The ubiquitin conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function is reliant on interactions with partner proteins and disruption of these is an effective way of modulating activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity within the Ube2d family of E2 enzymes. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV blocks a critical non-covalent ubiquitin binding site on the E2 enzyme. The discovery of novel protein-based ubiquitin derivatives that inhibit protein-protein interactions is an important step towards discovery of small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes.