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Abstract 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-Ub2combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2are 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-Ub2modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.
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Mode of Targeting to the Proteasome Determines GFP Fate
The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome’s ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub 4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub 4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates while the Ub 4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.
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- PAR ID:
- 10191518
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- jbc.RA120.015235
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1074/jbc.RA120.015235
