Genetically Encoded Crosslinking Enables Identification of Multivalent Ubiquitin‐Deubiquitylating Enzyme Interactions

Abstract

Ubiquitin (Ub) proteoforms control nearly every aspect of eukaryotic cell biology through their diversity. Inspired by the widely used Ub C-terminal electrophiles (UbÀ E), here we report the identification of multivalent binding of Ub with deubiquitylating enzymes (Dubs) using genetic code expansion (GCE) and crosslinking mass spectrometry. While the UbÀ Es only gather structural information with the S1 Dub sites, we demonstrate that GCE of Ub with p-benzoyl-L-phenylalanine enables identification of interaction modes beyond the S1 site with a panel of Dubs of both eukaryotic and prokaryotic origin. Collectively, this represents the next generation of Ub-based affinity probes with a unique ability to unravel Ub interaction landscapes beyond what is afforded by cysteine-based chemistries.

Reading and Writing the Ubiquitin Code Using Genetic Code Expansion

“…Inspired by the covalent Ub C‐terminal electrophiles in Ub vinyl methyl ester (VME), Ub vinyl sulfone (VS), and Ub propargyl, Das and colleagues functionalized Ub in the Thr9 position with Bpa to probe Dub interactions [95] . While these cysteine‐reactive monoUb probes have served as indispensable proteomics and structural probes, the endogenous substrate of Dubs is thought to largely be polyUb chains and/or ubiquitinated substrates, suggesting their lack in profiling multivalent Ub‐Dub interactions.…”