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Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication, but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here we report the crystal structure of the papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that the two residues-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alpha-CoV PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating and proteolytic functions of PEDV PLP2 from a structural perspective. Importance Current challenges in CoVs include a comprehensive understanding of mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structure basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed: (i) three Ub-core interacting residues are essential for DUB function, (ii) two-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only the structural insights to the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.
Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication, but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here we report the crystal structure of the papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that the two residues-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alpha-CoV PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating and proteolytic functions of PEDV PLP2 from a structural perspective. Importance Current challenges in CoVs include a comprehensive understanding of mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structure basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed: (i) three Ub-core interacting residues are essential for DUB function, (ii) two-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only the structural insights to the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.
Background Protein–protein interactions play a crucial role in almost all cellular processes. Identifying interacting proteins reveals insight into living organisms and yields novel drug targets for disease treatment. Here, we present a publicly available, automated pipeline to predict genome-wide protein–protein interactions and produce high-quality multimeric structural models. Results Application of our method to the Human and Yeast genomes yield protein–protein interaction networks similar in quality to common experimental methods. We identified and modeled Human proteins likely to interact with the papain-like protease of SARS-CoV2’s non-structural protein 3. We also produced models of SARS-CoV2’s spike protein (S) interacting with myelin-oligodendrocyte glycoprotein receptor and dipeptidyl peptidase-4. Conclusions The presented method is capable of confidently identifying interactions while providing high-quality multimeric structural models for experimental validation. The interactome modeling pipeline is available at usegalaxy.org and usegalaxy.eu.
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