Fig. 4: The C-terminal tail of ISG15 is dominant for binding SARS-CoV-2 PLpro.

a Superposition of the ¹H,¹⁵N-HSQC spectra of ¹⁵N-ISG15-FL (0.1 mM) and the mixture of ¹⁵N-ISG15 (0.1 mM)/0.15 mM SARS-CoV-2 PLpro. Massive peak broadening and peak intensity loss indicate binding of ISG15-FL to PLpro. b Superposition of the ¹H,¹⁵N-HSQC spectra of ¹⁵N-ISG15-ΔC6 (0.1 mM) and the mixture of ¹⁵N-ISG15-ΔC6 (0.1 mM)/SARS-CoV-2 PLpro (0.15 mM). Negligible peak perturbation indicates minimum binding of ISG15-ΔC6 to PLpro. c ITC measurement for the binding of SARS-CoV-2 with ISG15-FL (black) and ISG15-ΔC6 (blue), respectively. d Structural analysis of the complex structure of ISG15/SARS-CoV-2 PLpro (PDB 6XA9 [https://doi.org/10.2210/pdb6xa9/pdb]) shows that the sidechains (black dashed lines) of D164 and E167 of SARS-CoV-2 PLpro are involved in the binding with the C-terminal tail of ISG15, other interactions are involved with the backbone (red dashed lines). e DUB cleavage assay using Ub-AMC, ISG15-AMC, or peptide-AMC shows that D164A and E167A mutants have impaired enzyme activity compared with wild-type SARS-CoV-2 PLpro. Data are presented as mean ± SEM, n = 3 independent experiments.