Abstract
Ubiquitylation is a post-translational modification that regulates almost all signalling pathways in mammalian cells and its disruption is associated with a range of diseases. Ubiquitylation results in attachment of a small globular protein, ubiquitin (Ub), to substrates. It is catalysed by a cascade of enzymes called E1, E2, and E3. Specificity is mediated at two levels. The E2 conjugating enzymes determine the function of Ub signal by building polyUb chains of different linkage, whereas the E3 ligases specify the substrate and enable its modification by ubiquitin.
Among E3s, homologues Arkadia and Ark2C, a RING subclass of E3 ligases, regulate substrates in multiple pathways, including TGF-β and BMP signalling. The ability of the Ark family to partner up with several E2s, including UbcH5b and Ubc13, enables generation of distinct polyubiquitin chains that have degradative and non-degradative functions. The E3 ligase activity of Arkadia and Ark2C is regulated by the recruitment of a secondary, regulatory Ub (UbR). This Ub molecule stabilises the donor ubiquitin (UbD) of the E2∼Ub conjugate for catalysis and enables substrate modification. Although this feature has been reported, the molecular mechanism that drives the stabilisation of UbD remained unclear. This thesis presents a crystal structure of an activated UbR-Ark2C:UbcH5b∼UbD complex, which revealed crucial UbR:UbD contacts, that help to explain how the UbD is activated by Ark2C. Biochemical validation of the UbR:UbD interface identified residues, that are important for Ub transfer.
To better understand how Ark2C selects between its E2 partners, UbcH5b and Ubc13, the structure of the Ark2C:Ubc13∼Ub:Mms2 complex was solved. This enabled a comparison of Ark2C:UbcH5b and Ark2C:Ubc13 interfaces, which revealed a high level of conservation in residues that are involved.
This work characterised two key features that appear to regulate the ubiquitylation activity and confer E2 specificity. A disordered β3-RING loop, adjacent to the Ark-RING domain was analysed. This motif is rich in charged amino acids and split into the basic and acidic region. These appear to have a distinct function. Activity and binding experiments suggest, that the basic residues of the β3-RING loop enhance UbR binding, whereas the acidic residues regulate the activity of UbcH5b. Additionally, an N-terminal extension, predicted to form a helical motif was examined. This extension, termed the YEELL motif, is conserved among four human RING E3s, and regulates activity of UbcH5b, but not Ubc13. The importance of the YEELL motif for substrate modification was also established.
These finding suggest, that RING E3 ligases might form complexes with multiple E2s through a conserved set of residues, however the E2-specific regulation might be maintained by features that are found outside the RING domain.