A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes

Abstract

The post-translational modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) represents one of the most important regulators in eukaryotic biology. Polymeric Ub/Ubl chains of distinct topologies control the activity, stability, interaction and localization of almost all cellular proteins and elicit a variety of biological outputs. Our ability to characterize the roles of distinct Ub/Ubl topologies and to identify enzymes and receptors that create, recognize and remove these modifications is however hampered by the difficulty to prepare them. Here we introduce a modular toolbox (Ubl-tools) that allows the stepwise assembly of Ub/Ubl chains in a flexible and user-defined manner facilitated by orthogonal sortase enzymes. We demonstrate the universality and applicability of Ubl-tools by generating distinctly linked Ub/Ubl hybrid chains, and investigate their role in DNA damage repair. Importantly, Ubl-tools guarantees straightforward access to target proteins, site-specifically modified with distinct homo- and heterotypic (including branched) Ub chains, providing a powerful approach for studying the functional impact of these complex modifications on cellular processes.

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

“… 10 A recent report employs orthogonal sortase variants and C-terminally mutated Ub monomers. 9 We hypothesized that the chimeric E1 could activate and load Ub oligomers to Ubc9, which would subsequently transfer the defined Ub chains onto a LACE-tag on a substrate protein ( Figure 3 d). This approach would make use of natural linkage-defined Ub chains, which are commercially available or can be readily prepared and purified to homogeneity using canonical linkage-specific E2s, E3s, and DUBs.…”

“…In this sense, it complements the method of ubiquitylation of proteins bearing a Lys(ε-Gly–Gly) residue recently reported by Lang and co-workers, which introduces two mutations at the C terminus of Ub to convert it to a sortase-compatible sequence. 8 , 9 …”

Challenges in the use of sortase and other peptide ligases for site-specific protein modification

“…Lang and coworkers. 149 The sortases and the peptidyl asparaginyl ligases such as Butelase and OaAEP1 show the greatest promise for future applications in protein engineering. While ligases generated from proteases such as subtiligase have potential, their requirement for ester and thioester substrates and low specificity makes their application in protein engineering more challenging.…”

“…They have subsequently expanded this work and demonstrated that the eSrtA(2A-9) and Srt5M are orthogonal enabling them to generate a range of complex tri-ubiquitin and mixed ubiquitin/SUMO scaffolds ( Scheme 6C ). 149 …”

“…148 (C) Extension of this approach to applications with multiple orthogonal sortases enables the synthesis of specific triubiquitin and diubiquitylated SUMO constructs using both internal and Nterminal labelling. 149 oligonucleotide as a linker, leading to a protein-DNA-protein product. In the same paper, one-pot orthogonal dual labelling was used to produce an antibody-probe conjugate (Scheme 7C).…”