De novo design of ligases for programmable site-specific protein ubiquitylation

De novo designed ubiquitin ligases using a cysteine-based catalytic triad achieved near-quantitative ubiquitin transfer to a selected lysine in a model peptide, from 48 tested designs.

• Why it matters: Site-specific ubiquitylated proteins are hard to generate, limiting study of individual modification events despite the large human E3 ligase repertoire.
• What they did: Using RFdiffusion3, the team designed ligases from scratch and screened 48 constructs for ubiquitylation of a model peptide, then applied the approach to PCNA K164.
• The result: For PCNA, screening 48 designs delivered a site-selective ligase and, with a single activity-increasing substitution plus E1-dependent donor activation, reached 90% conversion from unactivated ubiquitin and enabled di-, tri-, and tetraubiquitin chain transfer to K164.

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