Structural characterisation of a novel E3 ligase module and activity-based probe development for UBL E3 ligases

E3 ligase enzymes are key regulators of many aspects of eukaryotic biology via the promotion of the ubiquitylation and subsequent degradation of proteins. There have been over 700 E3s identified within the ubiquitin system with diverse mechanisms of E3 ligase activity, however, significantly less have been identified for ubiquitin-like proteins. Recently developed activity-based probes allowed for the identification of novel E3 ligase proteins involved in the ubiquitin system that carry out unconventional ubiquitylation. The development of UBL activity-based probes has the potential to identify novel E3 ligases in these systems via proteomic analysis. To date, seven UBR (ubiquitin ligase N-recognin) family E3s (UBR1-UBR7) have been identified with all members apart from UBR4 shown to contain an established catalytic E3 module. Using recently developed activity-based probes I identified a region within the large E3 ligase UBR4 responsible for its ability to function as an E3 ligase. The crystal structures identified a novel protein fold named the ‘hemi-RING’ which resembles half of a canonical RING domain, coordinating a single zinc ion responsible for E3 ligase activity. Subsequent work is required to further investigate the presence of a potential catalytic cysteine residue within UBR4 and to further understand UBR4’s mechanism of function.