PMID:24854014

Citation	Ratia, K, Kilianski, A, Baez-Santos, YM, Baker, SC and Mesecar, A (2014) Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like protease. PLoS Pathog. 10:e1004113
Abstract	Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a "ridge" region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.
Links	PubMed PMC4031219 Online version:10.1371/journal.ppat.1004113
Keywords	Amino Acid Sequence; Catalytic Domain; Coronavirus Infections/metabolism; Coronavirus Infections/virology; Crystallography, X-Ray; Cysteine Endopeptidases/chemistry; Cysteine Endopeptidases/genetics; Cysteine Endopeptidases/metabolism; Cytokines/metabolism; HEK293 Cells; Humans; Models, Molecular; Protein Conformation; Protein Processing, Post-Translational; SARS Virus/enzymology; SARS Virus/genetics; SARS Virus/metabolism; Substrate Specificity; Ubiquitin/metabolism; Ubiquitination/genetics; Ubiquitins/metabolism; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
CVHSA:R1AB	enables	GO:1990380: Lys48-specific deubiquitinase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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