Characterization of Deubiquitinase Catalytic State Using a Structure-Based Approach

The activity of deubiquitinases (DUBs) is tightly regulated in eukaryotes via various mechanisms. One of the regulatory strategies is substrate-induced catalytic triad rearrangement, where ubiquitin-binding helps the DUB adopt an active conformation for catalysis. The crystal structure of the apo form of such a DUB, when not bound to ubiquitin, reveals an inactive conformation of the catalytic residues, necessitating the structure of the ubiquitin-bound form to visualize the active state of the DUB. Comparing the apo and ubiquitin-bound structures reveals conformational changes leading to catalytic activation. To capture the deubiquitinase in its ubiquitin-bound form, a series of activity-based ubiquitin probes (Ub-ABPs) harboring C-terminal electrophiles were designed to react with the catalytic nucleophile of cysteine protease DUBs. The resulting covalently linked DUB-ubiquitin complex is amendable for structural studies to probe the DUB-ubiquitin interface and the potential conformational change of the DUB. Here, we present a detailed protocol for the generation and purification of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in complex with a Ub-ABP, ubiquitin-vinyl methyl ester (UbVME), and the subsequent structural analysis to characterize the catalytic state of the DUB.