• 2018-07
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  • 2019-10
  • How does an increase in E affinity for the


    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. A role for the E2–E3 interface in governing mono- versus polyubiquitination is not mutually exclusive with that of the E2 backside, which has previously been shown to mediate polyubiquitination through its ability to bind to Ub [21], [25], [33]. Brzovic and colleagues first demonstrated the importance of backside Ub binding by UBCH5 family E2s to processive auto-polyubiquitination of BRCA1 [25]. A recent study of UBCH5B–RNF38 suggested that backside Ub binding promotes polyubiquitination by stabilizing the E3–E2~Ub complex in catalytically competent conformation [41]. Conversely, low-affinity E3 binding, as observed here for the RNF4–RAD6B interaction and as has been reported for yeast Rad6 [32], [34], would be expected to have the converse effect and be less effective at stabilizing the E2~Ub conjugate. It has long been clear that a simple model of ubiquitination, whereby the E2 simply accepts the activated Ub from the E1 and assists in the E3-directed ubiquitination of substrate, significantly underestimates the crucial role played by E2 Methicillin sodium salt writing the Ub code. Numerous studies have demonstrated the important role of E2s in determining the topologically distinct Ub modifications that form the basis of that code [3], [9], [14]. While E2 family members clearly enlist a variety of approaches in regulating ubiquitination, their general ability to harness multiple binding interactions to dictate ubiquitination specificity and topology has arisen as a common mechanistic theme within the family [3], [20], [21], [25], [35], [37]. Our finding that the affinity of the RNF4 for its cognate E2 plays an important role in determining the topology and multiplicity of substrate ubiquitination demonstrates the importance of yet another E2 binding interaction in governing ubiquitination. Going forward, it will be important to ask whether the E2-RING interaction plays a role in governing substrate ubiquitination by other E2–E3 pairs. Additionally, further investigation into potential crosstalk between the E2-RING interface and other E2-binding interactions, such as backside Ub binding, could provide a clearer understanding of how E2 enzymes govern the nature of ubiquitination.
    Materials and Methods
    Acknowledgments We thank Mario Amzel, Joel Tolman, Michael Matunis, Jie Xiao, Albert Lau, Dan Leahy, Ananya Majumdar, Reuven Wiener, and Chris Berndsen for valuable feedback, advice, discussions, and assistance. This work is supported by a grant from the National Institute of General Medical Sciences (GM109102).
    Introduction Among the various post-translational modifications made to proteins, ubiquitination emerges as one of the best studied, affecting the spatiotemporal regulation of numerous proteins involved in key cellular process, such as the cell cycle [1], [2], [3], [4], transcription [5], [6], and apoptosis [7], among other functions. Ubiquitination involves the conjugation of ubiquitin (Ub) to a lysine residue on the substrate, and this mostly occurs via an E1, E2, and E3 enzymatic cascade [8], [9]. The E1-activating enzyme modifies and covalently links with the Ub molecule, binds to an E2-conjugating enzyme, and transfers the Ub to the E2 active-site cysteine residue. The E3‐ubiquitin ligases are then responsible for the final step of the cascade, where they bind specifically to the target and approximate the target with the E2~Ub conjugate to transfer Ub to the target lysine residue.