Fig. 3: NMR data showing PLpro^CoV-2 interactions with hISG15, K48-Ub₂, and monomeric Ub.

a–d Overlay of ¹H-¹⁵N NMR spectra of ¹⁵N-labeled a hISG15, b distal Ub in Ub₂, c proximal Ub in Ub₂, or d Ub₁, alone (blue) and with 1–1.25 (2 for Ub₁) molar equivalents of unlabeled PLpro^CoV-2-C111S (red). Signals of select residues are indicated. Inset in (d) illustrates the gradual shift of the G47 signal during titration. e–g Overlay of ¹H-¹⁵N NMR spectra of ¹⁵N-PLpro^CoV-2-C111S, Y171H alone (blue) and with 1.25–1.5 molar equivalents of unlabeled e hISG15, f Ub₂, or g eight molar equivalents of Ub₁ (red). Insets zoom on the region containing indole HN signals of tryptophans (W93 and W106) and HN of the imidazole ring of histidine H272; the bound signals are marked with an asterisk. h hISG15 residues with strong signal perturbations mapped (yellow) on our structure of PLpro^CoV-2:hISG15 complex. i Residues in the proximal and distal Ubs with strong signal perturbations mapped (yellow) on our structure of PLpro^CoV-2:K48-Ub₂ complex. j Competition for PLpro^CoV-2 binding between Ub₂ and hISG15. (Left) Representative ¹H-¹⁵N NMR signals of G47 in ¹⁵N-labeled proximal Ub of Ub₂ pre-mixed with unlabeled PLpro^CoV-2-C111S,Y171H (in 1:1.5 molar ratio) upon addition of unlabeled hISG15, for indicated values of hISG15:Ub₂ molar ratio. (Right) The intensity of the PLpro-bound signal of G47 as a function of [hISG15]:[Ub₂] (dots) and the predicted molar fraction of bound Ub₂ (line) based on the K_d1 values obtained in this work (Supplementary Table 1). The symbols depict normalized peak intensities extracted directly from the respective 2D NMR spectra and the error bars represent experimental uncertainties in intensities obtained by error propagation using the experimental noise measured over at least five different regions in the spectrum that do not contain protein signals. k SDS-PAGE gels showing the inhibitory effect of hISG15 on disassembly of K48-linked Ub₂ by PLpro^CoV-2 (right) and minimal effect (if any) of hISG15 on disassembly of K48-linked Ub₃ (left). The hISG15 and Ub₂ constructs used here all had G (G157 or G76) as the C-terminal residue. Cleavage assays were repeated at least two times with similar results. Source data are provided as a Source Data file.