Fig. 6: A cryptic S2 ubiquitin-binding site in USP53 and USP54 mediates efficient cleavage of K63-linked ubiquitin chains.

a, Structure of USP54 conjugated to K63-linked diubiquitin–PA. b, Interface of ubiquitin and USP54 S2 site. c, Sequence alignment of residues forming the S2 site in USP54 and USP53. Cys-x-x-Cys motifs at the tip of the fingers (blue), conserved residues (green), residues unique in USP53 and USP54 within the human USP family (box) and residues annotated in b (arrows) are highlighted. d, Gel-based polyubiquitin cleavage assays. K63-linked ubiquitin chains (2 µM) were incubated with WT USP54^21–369 or the S2 site mutant F161K (both at 300 nM). Substrate consumption was quantified by densitometry, normalized to initial intensities. Data are shown as the average ± s.d. of three independent replicates. e, Gel-based polyubiquitin cleavage assays of USP53, shown as in d. WT USP53^20–383 or the S2 site mutant Y160K was used (2 µM). f, Catalytic efficiencies of USP54 proteins obtained from fluorescence polarization assays (substrates shown in Fig. 2d). Raw data are shown in Extended Data Fig. 9d,e and the catalytic efficiencies of WT protein are repeated from Fig. 2g. Data are shown as the mean ± s.e.m. g, Catalytic efficiencies of USP53 proteins, analyzed as in f. Efficient catalysis of USP53 is dependent on its S2 site. h, Schematic of ubiquitin processing by DUBs. K63-linkage-directed deubiquitination by USP53 bridges canonical DUB categories. The structural mechanisms for polyubiquitin length and linkage specificity in DUBs are shown in Extended Data Fig. 10.

Source data