Modulation of Ube2R1 activity by a nanobody that binds near its N-terminus

Ube2R1 (Cdc34) is a K48-ubiquitin chain-specific ubiquitin-conjugating (E2) enzyme central to proteasomal degradation, yet the regulatory potential of its unique structural elements remains underexplored. Here, we report the isolation and biochemical characterization of a nanobody, VHH12R1, that binds selectively to the N-terminal extension of Ube2R1 with no detectable cross-reactivity to its paralog Ube2R2 or other E2s. Engagement of this N-terminal region by VHH12R1 transiently delays ubiquitin charging, promotes accumulation of stable mono-ubiquitin Ube2R1 conjugates, and markedly reduces self-directed polyubiquitination chain formation by Ube2R1 without impairing di-ubiquitin synthesis. Although Ube2R1 can catalyze ubiquitination of VHH12R1 in the absence of an E3 ligase, this modification occurs independently of stable nanobody binding and is not required for modulation of Ube2R1 activity. Together, these findings support a model in which VHH12R1 selectively restricts processive self-elongation by Ube2R1 through engagement of its N-terminal extension, without broadly inhibiting ubiquitin transfer reactions. Our results reveal an unappreciates regulatory role for the Ube2R1 N-terminus in controlling catalytic outcomes and highlight nanobody-based approaches as precise tools to dissect E2 enzyme function.