A Genetically Encoded Electrophilic Lysine Derivative Enables Sortase-Mediated Assembly of SUMO Activity-Based Probes

Activity-based probes (ABPs) have become powerful tools for profiling enzymes that write and erase ubiquitin (Ub) and ubiquitin-like modifier (Ubl) signals. Extending this strategy to small ubiquitin-like modifier (SUMO)-specific proteases in defined substrate contexts remains challenging, because site-specific SUMO attachment must be combined with precise electrophile placement near the scissile isopeptide linkage. Here, we introduce a sortase-enabled chemoenzymatic platform for generating SUMO ABPs ranging from monoSUMO probes to native-like SUMO-substrate conjugates. The engineered sortase Srt2A ligates SUMO variants to glycine-bearing electrophiles, providing facile access to monoSUMO probes that trap deSUMOylases in vitro, in cellular lysates, and in living cells. To generate SUMO-substrate probes, we develop AzGVAisoK, a genetically encodable bifunctional lysine derivative containing both an azide-protected sortase handle and a vinyl amide electrophile. An engineered pyrrolysyl-tRNA synthetase/tRNA pair enables its site-specific incorporation into target proteins. Subsequent on-protein Staudinger reduction and sortase-mediated SUMOylation furnish defined SUMO-substrate ABPs under mild aqueous conditions. Applying this platform to PCNA and K11-linked diSUMO conjugates revealed distinct deSUMOylase trapping profiles governed by SUMO paralog and acceptor-substrate contexts. This work establishes a modular route to native-like SUMO probes and provides a general strategy for interrogating context-dependent enzyme recognition in Ubl signaling.