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

Abstract

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.

Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase

“…In factin contrast to sortase-generated diUbs Oa AEP1-generated diUbs are cleaved by DUBs to a similar extent as diUbs bearing a wt-linker, confirming their functional and structural resemblance to endogenous Ub/Ubl-conjugates. In this sense, Oa AEP1-mediated ubiquitylation complements the approach recently reported by Bode and co-workers that requires the introduction of up to three point mutations into the POI to make it a substrate for Ubc9-mediated ubiquitylation. , Ultimately, we envision that Oa AEP1-mediated generation of Ub and Ubl conjugates may be combined with sortylation to further extend our recently developed approach Ubl-tools (Ubl-topologies via orthogonal sortylation) as a modular and robust tool for accessing defined Ub/Ubl chains where we can place DUB-resistant and DUB-susceptible linkages at defined positions.…”

“…In this sense, OaAEP1-mediated ubiquitylation complements the approach recently reported by Bode and co-workers that requires the introduction of up to three point mutations into the POI to make it a substrate for Ubc9-mediated ubiquitylation. 16,46 Ultimately, we envision that OaAEP1mediated generation of Ub and Ubl conjugates may be combined with sortylation to further extend our recently developed approach Ubl-tools (Ubl-topologies via orthogonal sortylation) 29…”

Polyalanine disease mutations impair UBA6-dependent ubiquitination

“…2 ). Moreover, a recent study demonstrated that replacing the SCCH domain of UBA1 with the one of UBA2 allows the engineered UBA1 to load ubiquitin onto the E2 of SUMO (UBC9) 23 . Consequently, we assumed that the SCCH domain of UBA6 may hold specificity to USE1 and thus mutating the above-mentioned positive charged residues in UBA6 may affect the binding to USE1.…”

Post‐Assembly Modification of Protein Cages by Ubc9‐Mediated Lysine Acylation

“…In terms of reaction setup, the in vitro procedure allowed us to introduce a variety of synthetic and recombinant thioesters under controlled conditions. We have recently reported an engineered E1 enzyme that can activate and load ubiquitin onto Ubc9 in the cytoplasm of E. coli [28] . In the future, this E1 system could conceivably be used with an evolved Ubc9 variant to activate and conjugate desired peptide and protein moieties to cages in cellulo .…”

“…We have recently reported an engineered E1 enzyme that can activate and load ubiquitin onto Ubc9 in the cytoplasm of E. coli . [28] In the future, this E1 system could conceivably be used with an evolved Ubc9 variant to activate and conjugate desired peptide and protein moieties to cages in cellulo .…”