Native mass spectrometry reveals binding interactions of SARS-CoV-2 PLpro with inhibitors and ISG15

08 November 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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

Keywords

native mass spectrometry
SARS-CoV-2
variable temperature ESI
ultraviolet photodissociation
Ligand binding

Supplementary materials

Title
Description
Actions
Title
All PLpro SI
Description
Sequences, masses, and raw mass spectra for proteins, protein complexes, and inhibitors, sequence alignment for all PLpro proteins, UVPD data and identified fragment ions, and van’t Hoff plots.
Actions

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