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bioRxiv · 10.1101/2025.09.22.676137

Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W

Schnacke, P.·Fottner, M.·Kvasha, D.·Fabian, W.·Lang, K.
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Abstract

Deciphering the ubiquitin code requires homogenous, site-specifically ubiquitylated proteins, yet access to such conjugates remains a major challenge. Existing approaches are often constrained by low yields, harsh reaction conditions, engineered recognition motifs or non-native linkage architectures. Here, we present UbyW (Ubiquitylation by UBE2W), a programmable platform for site-specific ubiquitylation that repurposes the E2 enzyme UBE2W to target genetically encoded isopeptidic neo-N-termini. UbyW enables efficient generation of near-native Ub-protein conjugates across diverse protein substrates, including endogenous ubiquitylation sites within folded domains, and can be implemented through a reconstituted intracellular cascade in Escherichia coli for streamlined high-yield production. The platform further enables installation of chemical functionalities adjacent to the isopeptidic linkage, including photocrosslinkers for capturing modification-dependent interactions. Using programmable probes targeting site-specific ubiquitylation of the small GTPase Ran, we identify USP15 as a cognate deubiquitylase and show that Ran K71 monoubiquitylation disrupts key Ran-cycle interactions.

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Schnacke, P., Fottner, M., Kvasha, D., Fabian, W., Lang, K.. 2025-09-22. Genetic Code Expansion Facilitates Programmable Ubiquitylation via UBE2W. https://doi.org/10.1101/2025.09.22.676137

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