A modular Uba1-nanobody fusion enables selective ubiquitin transfer to tagged E2 enzymes.

The ubiquitin-activating enzyme Uba1 initiates the ubiquitination cascade by activating ubiquitin and subsequently transferring it to a broad range of E2 conjugating enzymes via its C-terminal ubiquitin-fold domain. Tools to selectively redirect this transfer to defined E2s, and thus govern control of E2 enzyme usage, are limited. By replacing the ubiquitin-fold domain of Uba1 with the high-affinity nanobody VHH05, we create an engineered E1 enzyme (Uba1-VHH05) that selectively engages E2s fused to the 6e-tag, which is the epitope recognized by VHH05. This plug-and-play interface preserves native Uba1 catalytic activity while allowing ubiquitin loading to be directed toward user-defined E2s without altering other components of the cascade. We show that Uba1-VHH05 supports transfer of both WT ubiquitin and the activity-based probe Ub-Dha to a range of tagged E2s. This recapitulates endogenous in vitro activities, including polyubiquitin chain formation. This strategy enables precise dissection of E2-specific functions and offers a new tool to generate orthogonal ubiquitin cascades in vitro and ultimately in cells.