Determination of the Mechanisms of Substrate Specificity of SARS-CoV-2 Papain-like Protease Through Structural and Biochemical Studies

Abstract

The COVID-19 pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and despite advancements in antivirals and vaccine development, SARS-CoV-2 remains a virus of concern. An avenue worth exploring is targeting the viral papain-like protease (PLpro) and 3C-like protease (Mpro), but to date, only the latter has FDA-approved therapeutics. PLpro is an essential protease for viral replication, with a primary role in processing viral polypeptides and a secondary role as a deubiquitinating and deISGylating enzyme. PLpro’s removal of poly-ubiquitin and ISG15 from target proteins enables the virus to evade the cellular immune response. Structurally K48-linked di-ubiquitin (Ub2) consists of two ubiquitin units connected via an isopeptide bond, which can be processed by PLpro. ISG15 is structurally similar to di-ubiquitin, consisting of two ubiquitin-like domains connected by an uncleavable linker along the backbone. Exploring how PLpro discriminates between ISG15 and Ub2 binding will provide structural insights for drug design aimed at disrupting the processing of these tags. In this work, nuclear magnetic resonance (NMR) spectroscopy and a variety of biochemical techniques were used to investigate how PLpro recognizes and processes substrates. As a result of this work, PLpro has been shown to exhibit dual-domain recognition of ISG15 and Ub2 and prefers occupation of both S1 and S2 sites for effective cleavage of substrate from the target. PLpro also requires the free C-terminal tail of the substrate to enter the active site, thereby undergoing a conformational change that stabilizes interactions within the S1 site. This work further established that strong binding to PLpro may not be required for the protease to process the substrate. As a result of the work in this thesis, a deeper understanding of how PLpro structurally recognizes substrates is provided, offering a framework for developing therapeutics that more efficiently target PLpro activity. Likewise, this work contributes foundational insights into how other PLpro variants of current and future coronaviruses may interact with substrates.