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

Abstract

The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) genome is evolving as the viral pandemic continues its active phase around the world. The Papain-like protease (PLpro) is a domain of Nsp3 – a large multi-domain protein that is an essential component of the replication-transcription complex, making it a good therapeutic target. PLpro is a multi-functional protein encoded in coronaviruses that can cleave viral polyproteins, poly-ubiquitin and protective Interferon Stimulated Gene 15 product, ISG15, which mimics a head-to-tail linked ubiquitin (Ub) dimer. PLpro across coronavirus families showed divergent selectivity for recognition and cleavage of these protein substrates despite sequence conservation. However, it is not clear how sequence changes in SARS-CoV-2 PLpro alter its selectivity for substrates and what outcome this has on the pathogenesis of the virus. We show that SARS-CoV-2 PLpro preferentially binds ISG15 over Ub and K48-linked Ub2. We determined crystal structures of PLpro in complex with human K48-Ub2 and ISG15 revealing that dual domain recognition of ISG15 drives substrate selectivity over Ub and Ub2. We also characterized the PLpro substrate interactions using solution NMR, cross-linking mass spectrometry to support that ISG15 is recognized via two domains while Ub2 binds primarily through one Ub domain. Finally, energetic analysis of the binding interfaces between PLpro from SARS-CoV-1 and SARS-CoV-2 with ISG15 and Ub2 define the sequence determinants for how PLpros from different coronaviruses recognize two topologically distinct substrates and how evolution of the protease altered its substrate selectivity. Our work reveals how PLpro substrate selectivity may evolve in PLpro coronaviruses variants enabling design of more effective therapeutics.

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

“…Based on the structural data this might be explained by the smaller overlap of our compounds with natural substrates binding to the S2 site. In addition, with binding affinities of the compounds in the high micromolar range, competitive inhibition will be difficult to detect in our experiments, as the affinity for ISG15 is reported in the lower micromolar range and ubiquitin affinity is approximately 120 µM ( Fu et al, 2021 ; Osipiuk et al, 2021 ).…”

“…Indeed, our biochemical characterization confirms an inhibitory effect of H1 on ubiquitin cleavage by PLpro. At the same time H1 binding to the S1 site does not reduce ISG15 cleavage by PLpro, which might be explained by the reported important interaction of ISG15 with the S2 site of PLpro ( Klemm et al, 2020 ; Osipiuk et al, 2021 ). As the central role of E167 is not only reported for SARS-CoV-2 but also for SARS-CoV-1 at this site ( Békés et al, 2016 ), the structural features of H1 have the potential to inhibit the deubiquitinase activity of different betacoronaviruses.…”

“…The C-terminal domain of human ISG15 binds mainly to the thumb domain at the S1 site of PLpro and interacts with a different set of residues compared to the PLpro/ubiquitin complex ( Figure 2B ). The key interaction sites mediating the contacts between PLpro and ISG15 can be found around ISG15 residues W123 ISG15 and P130 ISG15 ( Fu et al, 2021 ; Osipiuk et al, 2021 ) ( Figure 4B ). Within PLpro an overall upward shift of 1.7 Å in the interacting helix α7 becomes visible that strengthens ISG15 binding.…”

“…The hydrazone H1 is binding directly at the center of the S1 substrate binding interface, interfering with residues R166/E167 which are highly important for substrate recognition. Mutations at E167, which forms mandatory interactions with both substrates, strongly reduce PLpro activity ( Fu et al, 2021 ; Osipiuk et al, 2021 ). Binding of H1 has likely a similar effect on E167 as these mutations.…”

“…Nevertheless the S2 site is highly important for substrate recognition and PLpro activity in general. As T1–T5 are binding to the S2 site at the interface of the Ubl and thumb domain, their thiosemicarbazide moieties interact with residues P77 and T75, which together with V66 are critical for the substrate preferences of SARS-CoV-2 ( Shin et al, 2020 ; Osipiuk et al, 2021 ). Any mutations of these residues vary the surface properties.…”