Final Activity Report Summary - E3 LIGASE REGULATION (Regulation of Cullin based E3 ligases by the ubiquitin-like protein Nedd8/Rub1p) In order for eukaryotic cells to survive, they need to be able to properly dispense proteins that would otherwise inhibit essential functions. In most cases, a cell degrades proteins with a large protease called the 26S proteasome. The 26S proteasome digests proteins into their constituent amino acids, which can afterwards be reused in the synthesis of new proteins. In order to achieve this goal the proteins to be digested are marked with a 'degradation tag' that consists of a small polypeptide called ubiquitin. Continuous rounds of ubiquitin ligation, also referred to as ubiquitination, lead to the formation of ubiquitin chains on the target protein. These chains are recognised by the 26S proteasome, which in a subsequent step hydrolyses the tagged substrate. Ubiquitin ligation to substrate proteins requires three enzymatic steps, which are executed by an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ubiquitin ligase enzyme. The E3 ligases ensure substrate specificity, as they directly bind to the substrate and facilitate the transfer of ubiquitin onto the protein. Given the large number of cellular proteins that are marked for degradation by ubiquitination, it is not surprising that there are also many different E3 ubiquitin ligases present in the cell. The multi-protein cullin complexes represent one important type of E3 ligases. Like other ubiquitin E3s, cullin ligases recruit the substrate protein and the E2 enzyme to facilitate ubiquitin transfer. The core of this multi-protein complex is formed by cullins, which are large, rigid proteins that act as scaffold for the recruitment of other proteins to the ligase. Furthermore, cullins are a target of regulation to ensure proper and timely E3 ligase activity. Curiously, this regulation is achieved by a protein conjugation event that is very similar to ubiquitination. This ubiquitin-like system attaches the small polypeptide Nedd8 to cullins, which activates the E3 ubiquitin ligase by initiating ligase assembly and facilitating the recruitment of E2 ubiquitin. Like ubiquitination, neddylation also utilises and E1 activating and an E2 conjugating enzyme. However, prior to our work, no E3 Nedd8 ligase was described. Using biochemical and genetic approaches we have now identified the E3 Nedd8 ligase, which we called Dcn1. The main objectives of this proposal were to further characterise Dcn1 and to determine whether it was the E3 ligase for Nedd8. We were able to show that Dcn1, as expected for an E3 ligase, directly bound to both cullins and the Nedd8 E2 enzyme. Furthermore, we identified the Dcn1 binding site on the cullin, which was in very close proximity to the site of neddylation. We localised the reciprocal binding site on Dcn1 to a very C-terminal region of the protein, which we termed the DAD patch. Furthermore, we showed that Dcn1 also directly bound to the Nedd8 E2 enzyme and, in analogy to what was known for many ubiquitin E3 ligases, we could show that the surface on the E2 that interacted with Dcn1 was the same surface that interacted with the Nedd8 E1 enzyme. Importantly, we solved the X-Ray crystal structure of Dcn1, so that we were able to know what the protein looked like at a molecular level. The structure was entirely alpha helical and composed of an N-terminal UBA ubiquitin binding domain, and a novel C-terminal domain, which we termed PONY domain (for 'potentiating neddylation'). As implied by the name, the critical neddylation activity resided in the PONY domain, while the UBA domain was dispensable for function. All our structural and functional analysis indicated that Dcn1 acted like an E3 ligase. We were able to show that this was indeed the case by demonstrating that bacterially expressed, recombinant Dcn1 was able to catalyse the neddylation of cullins in a purified in vitro system. Consequently, we met the main objective of our proposal in that we identified the missing E3 ligase in cullin neddylation and hence a critical player for the regulation of ubiquitination.