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Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)‐conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N‐terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix–turn–helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase‐Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3‐dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2‐E3‐mediated polyubiquitination. Synopsis: Ubiquitin (Ub)‐conjugating E2 enzymes control substrate ubiquitination by dictating the linkage specificity of polyubiquitin chains and interacting with E3 ligases via specific surfaces, but the contribution of additional surface regions is unclear. Here, variable ubiquitin interactions of the E2 helix‐turn‐helix (HTH) motif are found to differentially modulate polyubiquitination in diverse E2s. Phylogenetic analysis reveals the HTH motif as a variable region, yet harboring functionally important and conserved charged residues within the distinct E2 family members.Ubiquitin binding to the E2HTH can be repurposed by different E2s, such as cell cycle regulators UBE2R/CDC34 and UBE2S, and tune intrinsic E2 activity.The positively charged UBE2R‐HTH facilitates acceptor ubiquitin recruitment and efficient chain elongation by dictating the dynamics of the gating loop near the enzyme active site.The negatively charged UBE2S‐HTH interacts with the donor Ub, influences the "open" and "closed" states of the UBE2S~Ub conjugate, and enhances ubiquitination by the E3 APC/C by reducing the conformation space of the "open" states. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC 50 to cisplatin and whole-cell proteomics reveal an increased abundance of ~20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2D's central role in proteome management.