br Results br Discussion The Ube E

Results
Discussion The Ube2E group of ubiquitin-conjugating enzymes, classified as the Class III E2s owing to their N-terminal extensions, is considered as “hub” E2s due to their capability of interacting with a large number of E3 ligases [26], [33]. The “E3 interactome” of these E2s is second only to that of the Ube2Ds and shares a significant overlap. Presumably, the resemblance between the UBC domain of these two E2 BRD-K4477 (~86% similar) results in their similarity in choosing cognate E3s. However, unlike Ube2Ds, the activity of Ube2E E2s has been observed to be limited with most of the interacting E3s including BRCA1-BARD1 [27]. Our results demonstrate that the limited activity of Ube2E1 is a direct consequence of ubiquitination at its flexible N-terminal extension. Thus, either the deletion of the entire N-terminus or mutating the Lys residues in that region to Arg relieves the attenuated activity of Ube2E1. The only exception to this effect of the N-terminal extension was the partial enhancement observed with Ube2E1∆N20 . Upon further investigation, we found that Ube2E1∆N20, unlike Ube2E1wt or Ube2E1∆N46, was unstable and invariably contained a contaminating degradation product of smaller molecular weight matching the “core domain” due to non-specific proteolysis of this truncated E2 construct. Thus, we concluded that the partial enhancement of Ube2E1∆N20 activity resulted from the contaminating core domain. We also note that though the deletion 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.