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  • We also note that though the


    We also note that though the Spectinomycin hydrochloride hydrate of the N-terminus in Ube2E1∆N46 or the mutations in Ube2E16KtoR enhanced their activity compared to the wild type, it failed to match Ube2D2 (Fig. 1). We concluded this difference to be multi-factorial as none of the Ube2E1∆N46 point mutants involving “Ub back-binding site” or the E3 interacting region could individually match the activity of Ube2D2wt (Supplementary Fig. 10). Interestingly, we also observed that the intramolecular ubiquitination of Ube2E1 is a very slow process if the E2 is investigated in isolation (Fig. 2). This explains why most of the thio-ester formation assays, typically carried out for shorter time intervals, failed to detect Ube2E1 self-ubiquitination. Intriguingly however, the presence of an interacting E3 ligase, such as RNF4 or cIAP2, drastically enhances the self-ubiquitination of Ube2E1, thus progressively dampening the E2 activity (Fig. 4). Mechanistically, this E3-mediated enhancement could be attributed to E2~Ub thio-ester adopting a catalytically competent conformation upon binding to the E3, thus facilitating the Ub transfer [34], [35]. Our failure to identify any single lysine in the N-terminal extension of Ube2E1 as the Ub acceptor together with the intramolecular nature of the modification underscores the importance of the N-terminal flexibility. As it can be readily appreciated that the N-terminal Lys residues could come in close proximity to the active-site cysteine due to their flexible nature (Fig. 7) and any structuring of this region would have prevented such self-modification. Given this importance of the flexibility, we probed if mutating the Lys residues in the N-terminus led to any stable secondary structure formation by CD spectroscopy and also by limited proteolysis (Supplementary Fig. 7). The result shows that both the wild type and the mutant Ube2E1 behaved identically, ruling out any alteration in the structure of the extension or that of the UBC core. We also extended our observations with Ube2E1 to other members of Ube2E class of E2s to show that self-ubiquitination-mediated activity attenuation is preserved across the Ube2E family. Sequence analysis revealed that this preservation is a consequence of perfectly conserved lysines in the N-terminal extensions of all the three Ube2Es despite having significant differences in their lengths and the sequences. Thus, substituting all the N-terminal Lys residues with Arg in Ube2E2 and Ube2E3 alleviates progressive suppression of their activity and renders them to be equally active as the corresponding UBC cores similar to Ube2E1 (Fig. 6). The importance of N-terminal lysine residues in attenuating Ube2E activity also does not appear to be UBC domain specific as fusing the N-terminal extension of the Ube2E1 to Ube2D2 (Ube2D2-NE1) rendered the chimeric E2 to show limited activity alike Ube2E1wt as reported by Schumacher et al.[28] while fusing the N-terminus of the Ube2E16KtoR to Ube2D2 did not alter the activity of the resultant E2 enzyme (data not shown). Our observations indicate that the limited activity of Ube2Es observed in vitro occurs due to two distinct reasons. Firstly, the activated Ub molecule from the catalytic site cysteine is alternatively transferred onto the E2 itself, rather than the E3 or the substrate, thereby reducing the amount of ubiquitin transferred to the latter. Secondly, self-ubiquitination progressively reduces the E2 activity slowing down fresh rounds of ubiquitin transfer by the modified E2 from the activating E1 enzyme to the E3/substrate. We, however, hypothesize that the first reason is more relevant in vivo. Our hypothesis gains support from the fact that the deubiquitinase (DUb) Usp7 (ubiquitin-specific-processing protease 7) has been demonstrated to de-ubiquitinate Ube2E1 in cells and prevents its proteasomal degradation [36]. In either case, Usp7-mediated de-ubiquitination of Ube2E1 at least indicates that self-ubiquitination serves a regulatory role in vivo rather than controlling the E2 turnover. Interestingly, the putative Usp7 binding sequence [36] (P/AXXS, where X is any residue) is conserved in both Ube2E2 (P12STS15) and Ube2E3 (P13STS16), suggesting possible role of Usp7 in regulating all three members of the Ube2E family.