Fig. 4: A K63-linked diubiquitin–PA probe enabled crystallization of USP54 in complex with ubiquitin.
From: Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53
a, Schematic of the generation of a K63-linked diubiquitin probe, which was enzymatically assembled from ubiquitin K63R and ubiquitin–PA. IEC, ion-exchange chromatography. b, Schematic of catalytic DUB domains after reaction with ubiquitin–PA or diubiquitin–PA probes, illustrating ubiquitin engagement in samples used in c,d. S1′, S1 and S2 ubiquitin-binding sites are labeled. The active site cysteine is depicted as a star. c, Protein stability assessment. USP5320–368 and USP5421–369 were labeled with ubiquitin–PA or diubiquitin–PA probes and the stability of protein samples was analyzed by thermal shift analysis. Melting temperatures (Tm) are shown for technical replicates, indicating the contribution of the S2 site. d, Purified DUB samples. USP5421–369 and USP5421–369 conjugated to ubiquitin–PA and USP5421–369 conjugated to K63-linked diubiquitin–PA were analyzed by SDS–PAGE and Coomassie staining. e, Crystal structure of USP54 conjugated to K63-linked diubiquitin–PA. In the observed complex, two USP54 molecules (blue and red) engage two K63-linked diubiquitin–PA molecules (gold and wheat) crosswise. The two isopeptide bonds between the diubiquitin–PA molecules are highlighted and are shown as sticks. f, SEC–MALS experiment of the catalytic domain of USP5421–369 alone (blue), in complex with ubiquitin–PA (brown) or in complex with K63-linked diubiquitin–PA (yellow), demonstrating monomeric species in solution. g, Schematic depiction of the organization of USP54 conjugated to K63-linked diubiquitin–PA in the crystal and in solution.
