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  • br Results br Discussion Many studies have shown that


    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 Metaphit 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 systolic pressure 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]. How does an increase in E2 affinity for the RING domain favor higher activity and polyubiquitination by the E2? Binding of the RING domain to the E2 positions the donor Ub in the E2~Ub conjugate in a closed complex [16], [17], [18] that increases the reactivity of the thioester [60]. A higher affinity of the RING for the E2 would promote the closed E2~Ub complex and thus make the thioester more reactive. Indeed, we observed a correlation between the affinity of RNF4 for the E2 and the reactivity of the E2~Ub thioester to lysine discharge (Fig. 5). Formation of polyubiquitin chains, in which Ub itself is the substrate, may depend upon a more reactive E2~Ub conjugate as compared to monoubiquitination of other substrates. We cannot rule out the possibility that the mutant RAD6B enzymes, and UBCH5B, are allosterically activated by the RNF4 RING in a different manner than wild-type RAD6B. Either a larger set of favorable interactions or a longer E2–E3 complex lifetime could contribute to different allosteric activation of the E2, although how this would translate into different multiplicity or activity is unclear.