PNNL

Abstract

Ubiquitin proteoforms control nearly every aspect of eukaryotic cell biology. Its diversity bridges a large gap in our understanding of ubiquitin mediated signaling as it spans linkage specific polyubiquitylation of homo- and heterotypic chains, other post-translational modification coherent with ubiquitylation (such as phosphorylation and acetylation), and attachment to substrate protein. While the ‘writers’ of this ubiquitin code create this diversity in ubiquitin modification, deubiquitylating enzymes bridge this gap in substrate potential by rendering ubiquitylation reversible to maintain homeostasis. Inspired by the ubiquitin C-terminal electrophiles, here we genetically incorporate the photoactivatable amino acid p-benzoyl-L-phenylalanine into ubiquitin as an interaction probe and identify crosslinked peptides by LC-MS/MS to capture interacting interfaces of ubiquitin with deubiquitylating enzymes. Mapping these interfaces enabled the distinction between different interaction modes of deubiquitylating enzymes. Collectively, this type of probes represents the next generation of ubiquitin-based affinity probes with a ubique ability to unravel ubiquitin interaction landscapes beyond what is afforded by cysteine-based chemistries.