Fig. 6: Structural basis for specific inhibition of UCHL1.

a Average distance alignment and schematic representation of domain architecture of human UCH family deubiquitinases. Boundaries of bacterially expressed UCH catalytic domain constructs are given. ULD, UCH37-like domain. b Ubiquitin rhodamine cleavage assay of indicated recombinant UCH DUBs, preincubated with GK13S dilutions for 1 h. Data points are shown as mean ± standard error from N = 2–5 independent experiments, as indicated in the figure. Source data are provided as a Source Data file. c Superposition of UCHL1~GK13S with apo UCHL1 (pdb: 2ETL) and apo UCHL3 (pdb: 1UCH) and close-up view of the binding pocket. Labeled residues are shown as sticks. d Superposition of the equivalent to the compound binding pocket in apo UCHL3 and UCHL3~Ub-VME (pdb: 1XD3). The Ubiquitin C-terminus is shown in cartoon representation. Residues undergoing a conformational change upon Ubiquitin binding are indicated with arrows. e Indicated Flag-UCHL3 constructs with mutations introducing a GK13S binding site were overexpressed in HEK293 cells. Cells were treated with a compound where indicated, and GK13S-bound proteins were visualized by in-gel fluorescence. Uncropped versions of gels and blots are shown in the supplementary information. f Sequence alignment of human Ubiquitin C-terminal Hydrolase (UCH) family members. Secondary structure assignments are based on the UCHL1~GK13S structure. Black arrows indicate residues mutated in e. For a full sequence alignment, see Supplementary Fig. 10a. g Close-up view of the binding pocket of UCHL1~GK13S (left panel) and superposition with full-length UCHL5 (pdb: 3IHR), the catalytic domain of UCHL5 (pdb: 3RII) and UCHL5 in co-complex with an inhibitory fragment of INO80G (pdb: 4UF5) (from left to right).