Multimodal mechanism of action for the Cdc34 acidic loop: a case study for why ubiquitin-conjugating enzymes have loops and tails

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pKa of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.