Extended Data Fig. 3: An S2 site, but not an S3 site, underlies USP53 and USP54 polyubiquitin cleavage.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a. Time-resolved gel-based cleavage assays of K63-linked tetraubiquitin (2 µM) by USP5320-383 (4 µM, left) and USP5421-369 (300 nM, right). Cleavage activity was analyzed by SDS-PAGE and Coomassie staining. Uncropped versions of all gels are provided as Source Data. b. Schematic of expected products of a triubiquitin cleavage assay in which the reagent contains a non-cleavable, C-terminal fluorescent label. Ubiquitin binding sites in a DUB are shown, expected cleavage sites are indicated with arrows. The presence of an S2 site would lead to fluorescently labeled monoubiquitin, whereas the presence of an S2’ site would lead to fluorescently labeled diubiquitin. c. Gel-based cleavage assay with a FlAsH-based reagent. Native K63-linked triubiquitin chains, K63-linked triubiquitin with a C-terminal tetracysteine (TC) tag in the proximal Ubiquitin (Ub3-TC) and FlAsH-labeled triubiquitin (Ub3-TC-FlAsH, all at 3 µM) were incubated with USP5421-369 (300 nM) for indicated times. USP54 activity was impaired by the presence of the FlAsH dye. d. Schematic of the generation of the TAMRA-based, fluorescent K63-linked triubiquitin substrate. TMR, TAMRA. e. Gel-based cleavage assay with a TAMRA-based reagent. Equivalent assays as shown in panel c with K63-linked triubiquitin chains shown in panel d (3 µM substrate, 300 nM enzyme). f. Schematic of the generation of the TAMRA-based, fluorescent K63-linked tetraubiquitin substrate. g. Gel-based cleavage assay. Fluorescent K63-linked tetraubiquitin (3 µM) was incubated with USP5320-383 (4 µM) or USP5421-369 (300 nM). Cleavage activity was analyzed as in Fig. 2e. The equally formed fluorescent diubiquitin and monoubiquitin species demonstrate that neither USP53 nor USP54 possess S3 ubiquitin binding sites. h. Deconvoluted intact protein mass spectra of indicated reagents. Calculated and observed molecular weights are given in Dalton. i. Fluorescence polarization cleavage assays of USP5320-383 and USP5421-369 and indicated reagents. Catalytic efficiencies derived from these data are shown in Fig. 2g.
