Site-Specific Protein Ubiquitylation Using an Engineered, Chimeric E1 Activating Enzyme and E2 SUMO Conjugating Enzyme Ubc9

Ubiquitylation-the attachment of ubiquitin (Ub) to proteins in eukaryotic cells -involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics -due to a rate-limiting thioester loading step and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant (k(cat)/K-M) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono-and oligo-Ub to various target proteins in vitro. Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.

Automated summary

Ubiquitylation is a process that attaches ubiquitin to proteins in eukaryotic cells and involves multiple enzymes. We have developed a method to transfer ubiquitin specifically to target proteins, which is important for studying protein function.