Abstract
The covalent attachment of ubiquitin to substrate proteins (ubiquitylation) is a post-translational modification that has major roles in regulating protein turnover and cell signalling. An E1-E2-E3 enzyme cascade tightly controls this process, but the nature of ubiquitin modification is highly variable. Single ubiquitin moieties or polyubiquitin chains of eight different linkages can be attached to proteins. The E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases have critical roles in determining both the substrate and the type of ubiquitin modification.
A large family of E3 ubiquitin ligases that contain both substrate recruitment and RING domains confer specificity within the ubiquitylation cascade. To activate ubiquitin transfer, RING domains bind an E2~ubiquitin conjugate and stabilise the ubiquitin moiety in a defined conformation that primes the active site on the E2 for nucleophilic attack. However, from the few examples that have been characterised, it is already clear that mechanisms used to achieve this vary between dimeric, monomeric and multicomponent E3 enzymes.
The RING E3 ligase, Arkadia (RNF111), is a key component of TGF- signalling and DNA damage-response pathways. Arkadia contains three SUMO interacting motifs (SIMs) and is classified as a SUMO-targeted ubiquitin E3 ligase (STUbL) because the SIMs enable it to target proteins that have previously been modified by SUMO for ubiquitylation. Two related proteins, Ark2C (RNF165) and Ark2N (ARKL1) retain high sequence homology with the RING- and SIM-containing portions of full-length Arkadia but exist as two separate polypeptides. The high conservation of the RING domains suggests that these two proteins may promote ubiquitin transfer in a similar manner.
Structural and biochemical analyses of the Arkadia and Ark2C RING domains were performed to investigate mechanisms underpinning their E3 ligase activity. The Arkadia and Ark2C RING domains were found to have very comparable properties in solution. For example, both RING domains are monomeric and both demonstrate similar E3 ligase activity in conjunction with the UbcH5b E2 as well as the Ubc13-Mms2 E2 complex. The structure of the Ark2C RING domain was solved and it was noted that regions of secondary structure were highly conserved in the Arkadia RING, thus the Arkadia RING is likely to have a comparable structure. For these reasons the Arkadia and Ark2C RING domains are commonly referred to as Ark-like RING domains.
Remarkably, the Ark-like RING domains were found to bind free ubiquitin with an affinity comparable to other dedicated ubiquitin-binding domains. A previously uncharacterised docking site on the -sheet of the RING domain facilitated this interaction. This identified the Ark-like RING domains as bona fide ubiquitin-binding domains. Further structural modelling and biochemical assays showed that the Arkadia RING domain and the non-covalently bound ubiquitin molecule co-ordinately stabilise the E2 conjugated ubiquitin (donor ubiquitin) in the ‘closed’ conformation so that ubiquitin transfer is promoted. These studies revealed a new role for the RING domain and provide insight into a new ubiquitin-dependent mechanism used by monomeric RING domains to activate ubiquitin transfer. This study also suggests how substrates that have been monoubiquitylated could be favoured for further ubiquitylation.