Fig. 5: Catalytic activity of USP53 and USP54 depends on a unique Cys loop.

a, Solution arrangement of USP54 conjugated to K63-linked diubiquitin–PA. Cartoon representation depicting USP54^21–369 (gray) with ubiquitin moieties bound in its S1 (wheat) and S2 (gold) binding sites. Zinc atoms are shown as gray spheres. b, Close-up view of the catalytic triad. Indicated distances are visualized by dotted lines. c, Sequence alignment of residues forming BL2 in USP DUBs. Catalytic histidines are colored red, while conserved residues are colored green. Residues or gaps in USP54 and USP53, which are unique across the entire human USP family, are highlighted with a box. Residues strongly conserved in the USP DUB family, which led to the initial mischaracterization of USP53 and USP54 as inactive, are highlighted in bold. Secondary-structure elements and numbering are according to USP54. d, Left, close-up view of BL1 and BL2 of USP54. Right, superposition with the corresponding residues in USP2 (PDB 2IBI, yellow) and USP14 (PDB 2AYO, purple), illustrating the atypical shortening of both loops in USP54. e, Left, close-up view of the unique loop of USP54 close to the catalytic cysteine. Right, superposition of the Cys loop with the corresponding residues in USP2 (yellow) and USP12 (PDB 5L8W, magenta). f, Sequence alignment of residues forming the Cys loop in USP DUBs (annotation as in c). Sequences of USP53 and USP54 constructs used to study the Cys loop are shown below with changes marked in violet. g, Gel-based ubiquitin chain cleavage assay. K63-linked triubiquitin chains (3 µM) were incubated with USP53^20–383 (3 µM) or USP54^21–369 (300 nM) as either WT or Cys loop mutant (labeled as LGNT) (sequences in f) for the indicated time points. Cleavage activity was analyzed by SDS–PAGE and Coomassie staining.

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