The structure of the HOIP
The structure of the HOIP RBR–LDD module bound to UbcH5~Ub reveals insights into an activated HOIP component . Contacts between the E2~Ub and the non-cognate RBR module in the crystal suggest how a structure in which the active sites of the E2 and the E3 are in close proximity might look. In this complex, the E2 binds to RING1 and the hydrophobic surface of ~Ub exposed in the open E2~Ub contacts the IBR–RING2 linker, bringing RING2 close to the E2 (Fig. 4B). For these contacts to be made by the cognate HOIP chain, other contacts involving the IBR will have to be disrupted. Finally, comparison of the HOIP RBR–LDD/E2~Ub and the RING2–LDD~Ub/Ubacceptor complex structures reveals that prior to the formation of the RING2~Ub species, the Ub moiety of E2~Ub bound to RING1 resides at the same RING2 site as the acceptor Ub that is required for Ub chain formation , . This observation seems to imply that Ub transfer from the E2~Ub onto the E3 active site must occur before the substrate (acceptor Ub) can bind in a manner that poises it for the ultimate reaction. There is currently no evidence whether a similar order of events is shared by other RBR E3s.
Concluding remarks While the last to be identified as a mechanistic class of E3 ligases, research on RBR E3s has provided important insights in a relatively short time frame. The main conceptual framework is in place: Important and fundamental questions remain:
Introduction Protein ubiquitination is a highly controlled enzymatic process involving formation of an isopeptide bond between the Gly76 of ubiquitin and a lysine residue of the target substrate. This multistep enzymatic process is carried out by the actions of ubiquitin-activation kras-pdeδ inhibitor (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) , . E1s are involved in a complex ATP-dependent mechanism initiating the ubiquitination process. In this step, E1s form a ternary complex consisting of E1-ubiquitin thioester bonded with ubiquitin-AMP. This complex achieves transthiolation for the downstream conjugation of ubiquitin. E2s are responsible for transferring the ubiquitin to substrate proteins and function together with E3 ligases. E3s play an extremely critical role in the recruitment of ubiquitin-E2 complexes, recognition of target proteins, and transfer of the activated ubiquitin. The selectivity of the protein ubiquitination by target proteins depends on the specificity of the E3 ligases. In addition, E3 ligases determine if the substrate is to be poly-ubiquitinated and whether or not it is degraded . Based on the binding between protein interaction and E2s, as well as the substrates, there are four major classes of ubiquitin ligase. These are (i) the homologous to E6-AP carboxyl terminus (HECT) domain-containing E3s, (ii) the really interesting new gene (Ring) finger domain-containing E3s, (iii) the U box E3s, and (iv) the multisubunit E3 complex that contains a RING finger protein and an adaptor . Protein ubiquitination has been investigated for many years and several known points of intervention and appropriate agents, especially the E3 ligases, have been identified. The E3 ligases control the selectivity of the substrate proteins and are responsible for the recruitment of specific substrate proteins that will be tagged with ubiquitins. Hence, selective inhibition of E3s may directly target an aberrant signaling pathway in several diseases such as cancer and autoimmune diseases . The identification of substrate binding surface and mediated ubiquitination activity provides significant evidence supporting the development of small molecule E3 inhibitors (Fig. 1) . But in view of the overall protein subunit arrangements combined with the need for small molecule inhibitors to bind and disrupt protein interfaces or otherwise affect ligase activity allosterically, considerable difficulties remain and relatively few compounds have been entered into clinical trials. Interestingly, most of the compounds developed as inhibitors, such as compounds 1-5, target the RING domain-E3 ligases rather than the HECT domain-E3 ligases. The involvement of HECT domain-E3 ligases in biological processes such as neuritogenesis and their emergence as crucial regulators in cancer development has happened only in the past decade , .