Extended Data Fig. 1: Characterization of Lbpro ubiquitin cleavage.
From: Insights into ubiquitin chain architecture using Ub-clipping
a, Representative raw spectrum of Lbpro-treated Lys48-linked diubiquitin analysed by electrospray ionization MS. Two species arise owing to internal cleavage of ubiquitin after Arg74. One scan is shown from experiments performed in technical triplicate. b, After 24 h of Lbpro treatment, diubiquitin was further supplemented with fresh Lbpro and incubated for an additional 24 h. There are no changes in the intensities of ubiquitin bands, which suggests that Lbpro products are stable. Lys27 diubiquitin in this panel and in Fig. 1a was generated chemically from synthetically produced ubiquitin and was refolded; this generates a variable fraction of substrate that cannot be hydrolysed by deubiquitinases (DUBs), leading to apparent lower activity due to incomplete hydrolysis. Diubiquitin cleavage assays were performed independently in duplicate. c, Model of ubiquitin cleavage by Lbpro. Ubiquitin (green) was modelled on the basis of the crystal structure of Lbpro (blue) bound to the C-terminal domain of ISG15 (PDB: 6FFA19). A close-up view shows the C terminus of ubiquitin placed across the active site, enabling cleavage between Arg74 and Gly75.
