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  • One of the most fundamental distinctions between Ub signals

    2020-11-20

    One of the most fundamental distinctions between Ub signals is substrate monoubiquitination versus polyubiquitination. With the exception of the E2, UBE2W, which represents a special case because it mk 801 sale only ubiquitinates the flexible N-termini of substrates [5], [6], [20], most examples of monoubiquitination studied to date involve a role for the E3 ligase in either suppressing intrinsic polyubiquitination activity of the E2 [21] or directing the E2 to a preferred target lysine in the substrate [22], [23]. The E3 ligase APC/C is capable of driving both the direct multi-monoubiquitination of substrate in conjunction with UBE2C and the extension of polyubiquitin chains on substrate with UBE2S, by interacting with each E2 using two distinct binding architectures [24]. UBCH5C contains a non-covalent ubiquitin-binding site on its “backside”, distal from the active site, which mediates polyubiquitination as assayed in E3 autoubiquitination assays [25]. However, the Bmi1–RING1 E3 complex targets UBCH5C to monoubiquitinate histone H2A–Lys119 [26], [27], [28] by juxtaposing the UBCH5C~Ub thioester and the target lysine [22], [23]. The E2, RAD6B, can synthesize polyubiquitin chains on its own in vitro[21], [29] but monoubiquitinates PCNA together with the E3, RAD18 [30], [31]. RAD18 suppresses intrinsic RAD6B polyubiquitination activity through a special domain called the R6BD, which binds to the RAD6B backside and suppresses its polyubiquitination activity [21]. Yeast Rad6 also has intrinsic polyubiquitinating activity [32] that is mediated in part through backside interactions [33]. Rad6 monoubiquitinates histone H2B–Lys123 in conjunction with the E3 ligase, Bre1 [32]. Although Bre1, like human RAD18, contains a special domain that contacts the Rad6 backside, the Bre1 RING domain alone is sufficient to direct the monoubiquitination of nucleosomal H2B [34], potentially through substrate targeting as recently observed in the mk 801 sale structure of Bmi1–RING1/UBCH5C bound to a nucleosome [23]. While backside interactions and direct targeting of the E2 to its substrate are clearly important in governing mono- versus polyubiquitination, there is evidence that the molecular interface between the E2 and the RING domain may also govern substrate ubiquitination [35]. Indeed, a screen for mutants of the U-box E3, UBE4B, which enhanced the auto-polyubiquitination of UBE4B in conjunction with the E2, UBCH5C, yielded a subclass of activating point mutations in UBE4B that increased its affinity for the E2 [36]. The activating mutations in UBE4B, while increasing the rate at which polyubiquitin accumulated, did not impact the multiplicity of autoubiquitination. Interestingly, whether the E2 enzyme, UBCM2 (UBE2E3), mono- or polyubiquitinates a RING E3 partner in autoubiquitination assays correlates with the ability of glutathione S-transferase (GST)-tagged UBCM2 to interact with the E3 in a pull-down assay: GST–UBCM2 pulls down AO7T, which is polyubiquitinated in conjunction with UBCM2, but not BD/BC, which is monoubiquitinated in conjunction with UBCM2 [37]. Since the E3 is also a substrate in these reactions, it is not possible to separate the potential contributions of substrate versus E3 affinity to the observed differences in ubiquitination. While a number of studies have helped elucidate the principles of target amino group specificity, UBL selectivity, and Ub linkage specificity [3], [4], [9], [10], [14], [19], [20], [23], [24], [33], [34], [37], [38], [39], [40], [41], the role of the canonical E2-RING interface in governing substrate ubiquitination is less well-understood. We therefore investigated how the differences in E2–E3 RING interactions affect substrate ubiquitination. In order to separate the role of interactions between the RING domain and the E2 versus the interactions between the charged E2 and the substrate, we utilized a system in which we could monitor the ubiquitination of a substrate other than the E3 ligase itself. RING finger protein 4 (RNF4) is a compact 190-residue E3 ligase that belongs to the SUMO-targeted Ub ligase subfamily and directs the ubiquitination of polySUMO chains [42], [43]. RNF4 contains a C-terminal RING domain that binds the E2 and N-terminal SUMO-interacting motifs that bind to the polySUMO substrate [44], [45], [46]. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and robustly polyubiquitinates the substrate together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases [46], [47], [48], [49]. We find that the ubiquitinating activities of RAD6B and UBCH5B in concert with RNF4 are governed by interactions between the E2 and the RNF4 RING domain. By reengineering the RAD6B RING-binding surface to resemble that of UBCH5B, we transformed RAD6B into a UBCH5B-like E2 that polyubiquitinates polySUMO in the presence of RNF4. The switch from weak monoubiquitinating activity to robust polyubiquitinating activity correlates with increased affinity of the E2 for RNF4. Our results shed new light on the characteristics of E2-RING interactions that govern the activity and nature of substrate ubiquitination.