Abstract
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.
Highlights
Ubiquitin-mediated proteolysis is the principal mechanism for regulating protein half-lives in cells
The amount of substrate and product were quantified and used to calculate the percentage of substrates that were modified by one or more ubiquitins. This is equal to the sum of the intensities of all bands above the substrate band divided by the total intensity in the lane
Two Conserved Acidic Residues in the Human Cdc34 Acidic Loop Promote the Interaction of Cdc34 with Ubiquitin Ligase SCF—Many enzymes contain flexible loops near the active site that participate in catalysis
Summary
Ubiquitin-mediated proteolysis is the principal mechanism for regulating protein half-lives in cells. Results: Cdc promotes ubiquitin chain assembly onto protein substrates and contains an essential acidic loop near the active site. 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 Cdc which greatly contributes to Cdc catalysis These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis. All E2s share a common catalytic domain, Cdc contains two molecular add-ons including an acidic tail appended to the C terminus of the catalytic domain (18 –20) as well as a 12-residue insertion near the active site, commonly referred to as the acidic loop [21]. Soon after the discovery of Cdc, it was determined that the acidic tail promotes Cdc interaction with the SCF ubiquitin ligase, the archetypal CRL
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