Native Mass\nSpectrometry Reveals Binding Interactions\nof SARS-CoV‑2 PLpro with Inhibitors and Cellular Targets

Here\nwe used native mass spectrometry (native MS) to probe a SARS-CoV\nprotease, PLpro, which plays critical roles in coronavirus disease\nby affecting viral protein production and antagonizing host antiviral\nresponses. Ultraviolet photodissociation (UVPD) and variable temperature\nelectrospray ionization (vT ESI) were used to localize binding sites\nof PLpro inhibitors and revealed the stabilizing effects of inhibitors\non protein tertiary structure. We compared PLpro from SARS-CoV-1 and\nSARS-CoV-2 in terms of inhibitor and ISG15 interactions to discern\npossible differences in protease function. A PLpro mutant lacking\na single cysteine was used to localize inhibitor binding, and thermodynamic\nmeasurements revealed that inhibitor PR-619 stabilized the folded\nPLpro structure. These results will inform further development of\nPLpro as a therapeutic target against SARS-CoV-2 and other emerging\ncoronaviruses.