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  • While a number of studies have helped elucidate the


    While a number of studies have helped elucidate the principles of target amino group specificity, UBL selectivity, and Ub Pirarubicin 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 Pirarubicin 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.
    Discussion Many studies have shown that intrinsic properties of the E2 enzyme govern the nature of the Ub modification, including both the multiplicity of the Ub modification and the specific linkage type in the case of polyubiquitin chains [3], [4], [9], [14]. Whereas previous studies of both UBCH5 and RAD6 isoforms have pointed primarily to interactions with the so-called E2 backside in governing mono- versus polyubiquitination [21], [25], we find that the nature of the RING binding to the E2 plays a critical role in determining the overall activity and multiplicity of substrate ubiquitination. By making reciprocal mutations in the RING-binding surface of the monoubiquitinating E2, RAD6B, and the polyubiquitinating E2, UBCH5B (Fig. 2), we were able to convert RAD6B into a robust polyubiquitinating enzyme and UBCH5B into a weaker, monoubiquitinating enzyme (Fig. 3). Moreover, the polyubiquitinating activity correlated with the affinity of the E2 for the RNF4 RING domain, with increasing polyubiquitinating activity observed with increasing E2–E3 RING affinity. Our results are also consistent with the finding that tighter binding of the RING domain correlates with higher overall activity, as had been previously observed for gain-of-function mutations in the U-box E3, UBE4B [36]. Since the present study monitored substrate ubiquitination rather than autoubiquitination, we were able to separate the contribution of tighter interactions with the E2—and higher reactivity of the E2~Ub thioester (Fig. 5)—from tighter binding to the substrate. While our findings, to our knowledge, are the first to show a direct relationship between the affinity of the E2 for the RING domain and substrate mono- versus polyubiquitination, our results are consistent with observations made in a number of previous studies. As mentioned above, a possible correlation between the strength of E3–E2 interactions and mono- versus polyubiquitination was suggested by the observation that the multiplicity of E3 autoubiquitination in conjunction with the E2, UBCM2 (UBE2E3), correlated with whether E3 binding could be detected in pull-down assays with GST-tagged UBCM2 [37]. While the relative contribution of E3–E2 versus E3–substrate binding affinity could not be uncoupled because the E3 ligases in that study were both the substrate and the enzyme, our results point to a role for E3 affinity for the E2 enzyme as a determinant of mono- versus polyubiquitinating activity by UBCM2. In an earlier study, Haas and colleagues [58] described a similar phenomenon in an investigation of the E3-catalized substrate ubiquitination by the E2s, CDC34, and RAD6. While the identity of the E3 was unclear because Acceptor splicing site used a crude reticulocyte fraction containing E3 activity, they found that RAD6 exhibited a significantly higher E2–E3 Km than CDC34, an E2 known to polyubiquitinate substrates [59], suggesting that differences in E3 affinity might play a role in determining the differing substrate ubiquitination behavior of RAD6 and CDC34 [58]. A role for RING interactions in directing substrate monoubiquitination has also been suggested for yeast Rad6 [34], which monoubiquitinates nucleosomal histone H2B in conjunction with the RING E3 ligase, Bre1 [32]. The Bre1 RING also has very low affinity for Rad6 [32], [34]. A recent study found that a minimal domain comprising the Bre1 RING and a coiled-coil dimerization domain were sufficient to direct monoubiquitination of histone H2B [34]. Interestingly, directly fusing the coiled-coil RING fragment of Bre1 to Rad6 increased the rate of histone ubiquitination without significantly altering the overall pattern; the primary product was still monoubiquitinated H2B, with only slight increase of a secondary H2B ubiquitination site [34].