Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

Wydorski, Pawel M.; Osipiuk, Jerzy; Lanham, Benjamin T.; Tesar, Christine; Endres, Michael; Engle, Elizabeth; Jedrzejczak, Robert; Mullapudi, Vishruth; Michalska, Karolina; Fidelis, Krzysztof; Fushman, David; Joachimiak, Andrzej; Joachimiak, Lukasz A.

doi:10.6082/nrbfn-nx434

Published April 25, 2023 | Version v1

Journal article Open

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

1. University of Texas Southwestern Medical Center
2. University of Chicago
3. University of Maryland
4. University of California, Davis

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

Data availability

The structural datasets generated during the current study are available in the Protein Data Bank repository [https://www.rcsb.org/] under accession codes PDB ID: 7RBR for PLproCoV-2, C111S mutant, in complex with K48-Ub2; PDB ID: 7RBS for PLproCoV-2, C111S mutant, in complex with human ISG15; PDB ID: 7UV5 for PLproCoV-2, C111S,D286N mutant, in complex with K48-Ub2; PDB ID: 7S6P for human ISG15 alone, and PDB ID: 7S6O for K48-Ub2 alone. Diffraction images are available on Integrated Resource for Reproducibility in Macromolecular Crystallography repository set up by W. Minor laboratory [https://proteindiffraction.org] for PDB IDs 7RBR; 7RBS; 7UV5; 7S6P; 7S6O. NMR signal assignments for hISG15 used in this study are available under BMRB Entry ID 5658, assignments for Ub1 were based on BMRB Entry ID 17769, and for Ub2 on BMRB Entry IDs 30602 and 19406. All MST, processed cross-linking mass spectrometry, and ΔΔGcalc data are available in Source Data provided with this paper. Docking models used in this study are available on Zenodo under accession number 7768418. Raw MS data used for the XL-MS analysis are available in the MassIVE database under the accession number MSV000091075 and in the ProteomeXchange under the identifier PXD040822. Source data are provided with this paper.

All ΔΔGcalc calculations were performed using the flex_ddg protocol that is available on Kortemme Lab GitHub [https://github.com/Kortemme-Lab/flex_ddG_tutorial] using Rosetta v3.12 [https://www.rosettacommons.org/].

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