Mechanistic Studies of the Activation of Ubiquitin-Conjugating Enzymes by Ring-Type Ubiquitin Ligases

Ubiquitination, modification with ubiquitin, is a post-translational regulation of proteins in eukaryotes. Ubiquitin-activating enzymes (E1) activate ubiquitin and form thioester linkages with ubiquitin, which are then transferred onto ubiquitin-conjugating enzymes (E2). Ubiquitin-conjugating enzymes, with the help of ubiquitin ligases (E3) transfer ubiquitin onto ubiquitination targets to form isopeptide linkages between the targets and ubiquitin. The function of ubiquitin ligases in the ubiquitination process has not been clearly understood. Recently, it has been shown that ubiquitin ligases are modular proteins or protein complexes that bring together the ubiquitination targets and the E2 enzyme. In this study, we show that E3 enzymes without their target-binding domains are effective activators of the ubiquitination reaction. We demonstrate that E3 binding to E2 enzymes increases the rate of the breakdown of the E2-ubiquitin thioester bond. Using the Statistical Coupling Analysis, a pairwise residue covariation analysis, we observe that E2 enzymes have a set of residues that vary as a group throughout evolution. Extensive mutagenesis of the E2 enzyme UbcH5b, including its covarying residues, yield mutants that can accept ubiquitin, break the thioester linkage, bind E3 enzymes, but cannot be stimulated to release their ubiquitin thioesters in the presence of an E3 enzyme. We also show that thioester release measurements agree with polyubiquitination of physiological targets. We have structurally characterized our model E2 enzyme UbcH5b, it mutants, and its interaction with E3 enzymes. Taken together, our data strongly demonstrate that E3 enzymes are, apart from their scaffolding function, activators of E2 enzymes to ubiquitinate targets. We discuss possible physical mechanisms of this activation, and hypothesize a long-distance communication pathway between the E3-binding surface and the thioester-forming active site of E2 enzymes. We also report attempts to crystallize several E2 and E3 enzymes and their complexes.