Extended Data Fig. 9: A cryptic S2 site mediates catalytic activity on longer ubiquitin chains.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a. Purified samples of USP54 with mutated S2 site. USP5421-369 F161K, USP5421-369 F161K~Ub-PA and USP5421-369 F161K~diUb(K63)-PA were analyzed by SDS-PAGE and Coomassie staining. An uncropped version is provided as Source Data. b. Protein stability assessment of S2 site-mutated USP54. Stability of samples from panel a was analyzed by thermal shift analysis. Fluorescence raw data are shown. Melting temperatures (Tm) are plotted from technical replicates. c. Schematic of catalytic USP54 F161K DUB domains after reaction with Ub-PA or diUb-PA probes, illustrating ubiquitin engagement in samples used in panels a and b. S1’, S1 and S2 Ub binding sites are labelled. The mutated S2 site is depicted as a red star. d. Fluorescence polarization cleavage assays for USP5320-383 Y160K and indicated reagents. Fluorescence anisotropy was recorded over time and observed rate constants (kobs, shown as mean ± standard error) were plotted over enzyme concentrations. Values for WT protein were taken from Fig. 2g. e. Fluorescence polarization cleavage assays for USP5421-369 F161K and indicated reagents as described in panel d. f. Cartoon representation of USP54~diUb-PA, CYLD in complex with K63-linked diUb (PDB: 3WXG) and CYLD in complex with M1-linked diUb (PDB: 3WXF). A superposition illustrates features enabling K63-linkage recognition in both enzymes by distinct mechanisms. The catalytic ___domain of CYLD achieves dual K63 and M1 polyubiquitin cleavage specificity through a unique β-hairpin inserted into its catalytic ___domain. This insertion forms an S1’ ubiquitin binding site to recognize the Phe4 patch of the proximal ubiquitin (in both K63- and M1-linked chains) and positions the substrate for efficient catalysis. USP54 lacks an equivalent hairpin insertion.
