Chemical tools for structural studies of ubiquitin and ubiquitin-like deconjugating proteases

Attachment of ubiquitin and ubiquitin-like modifiers (UbLs) are reversible post-translational modification of proteins that regulate crucial cellular functions, ranging from protein homeostasis to the control of protein-protein interactions. In the cell genome, in addition to the high number of E3 ligases to control ubiquitin/UbL conjugation, a considerable number of deubiquitinases (DUBs) and Ubiquitin-like proteases (ULPs) are also encoded, indicating the essential role of the reversible deubiquitinating proteolytic activity. Most DUBs and ULPs are cysteine proteases, containing a nucleophilic cysteine in the active site that cleaves the isopeptide bond between ubiquitin and target substrate or between ubiquitin units in polymeric chains. Significant progress has been made in recent years regarding the identification of novel types of DUBs and ULPs, as well as in our understanding of their molecular mechanisms. This progress has been partially attributed to the development of specific chemical tools, such as Activity-Based Probes (ABPs), designed for studying DUB cysteine proteases. ABPs mimic enzymatic substrates and, in an enzyme-catalyzed reaction manner, remain covalently attached, resembling an irreversible competitive inhibitor. The structures formed by ABPs in complex with enzymes provide valuable insights into catalytic mechanisms and the interactions between ubiquitin and DUBs/ULPs proteases. In this review we will summarize recent advancements in the use of ABPs to characterize the structures of DUBs/ULPs in complex with ubiquitin/UbLs. Additionally, we will present relevant examples of complex structures of DUBs with specific ABPs ubiquitin-linked chains.