Figure 1. Biophysical and structural characterisation of PLpro activity.

• A
  Cartoon of coronavirus PLpro activities. PLpro is encoded as one of various domains of the 1,900 amino acid non‐structural protein nsp3 and is thought to have three functions: (i) cleaving the viral polyprotein to generate mature nsp1, nsp2 and nsp3; (ii) hydrolysing ubiquitin chains important for inflammatory responses and (iii) removing interferon‐stimulated gene 15 (ISG15) modifications from proteins, reversing antiviral responses.
• B
  Schematic of ubiquitin‐binding sites in SARS PLpro, which binds Lys48‐triubiquitin via S2, S1 and S1′ ubiquitin‐binding sites. Hydrolysis occurs between ubiquitin molecules bound at S1 and S1′.
• C
  Time course analysis of triubiquitin (2 μM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie‐stained SDS–PAGE gel. Linkage‐specific cleavage of Lys48‐linked triubiquitin to di‐ and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015, 2016). See Appendix Fig S1B–D for gel‐based cleavage quantification.
• D
  Overview of the catalytic efficiencies of PLpro. A fluorescence polarisation (FP) assay was used to derive the catalytic efficiencies (k _cat/K _M) of PLpro for the depicted substrates. Catalytic efficiencies were calculated from data shown in Appendix Fig S1A, as described in Materials and Methods. Substrate preference is indicated by x‐fold activity relative to Ub‐TAMRA cleavage.
• E
  Crystal structure at 2.7 Å resolution of SARS2 PLpro with subdomains coloured in shades of blue, bound to ubiquitin propargylamine (Ub‐PA, orange). Catalytic triad residues are shown in ball‐and-stick representation, and a Zn ion is indicated as a grey sphere. Also see Appendix Fig S2 and Table 1.
• F
  Crystal structure at 2.9 Å resolution of SARS2 PLpro (blue) bound to ISG15 C‐terminal domain propargylamide (ISG^CTD‐PA, salmon). Also see Appendix Fig S2 and Table 1.

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