X-ray Structure and Enzymatic Activity Profile of a Core Papain-like Protease of MERS Coronavirus with utility for structure-based drug design

Abstract

Ubiquitin-like domain 2 (Ubl2) is immediately adjacent to the N-terminus of the papain-like protease (PLpro) domain in coronavirus polyproteins, and it may play a critical role in protease regulation and stability as well as in viral infection. However, our recent cellular studies reveal that removing the Ubl2 domain from MERS PLpro has no effect on its ability to process the viral polyprotein or act as an interferon antagonist, which involves deubiquitinating and deISGylating cellular proteins. Here, we test the hypothesis that the Ubl2 domain is not required for the catalytic function of MERS PLpro in vitro. The X-ray structure of MERS PLpro-∆Ubl2 was determined to 1.9 Å and compared to PLpro containing the N-terminal Ubl2 domain. While the structures were nearly identical, the PLpro-∆Ubl2 enzyme revealed the intact structure of the substrate-binding loop. Moreover, PLpro-∆Ubl2 catalysis against different substrates and a purported inhibitor revealed no differences in catalytic efficiency, substrate specificity, and inhibition. Further, no changes in thermal stability were observed between enzymes. We conclude that the catalytic core of MERS PLpro, i.e. without the Ubl2 domain, is sufficient for catalysis and stability in vitro with utility to evaluate potential inhibitors as a platform for structure-based drug design.

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…In contrast to the variable relative orientations of the Ubl domains and the catalytic domain of USP7, the Ubl2 domain is anchored to the CoV PL2 pro by two salt-bridges in MERS-CoV and SARS-CoV (Lei et al, 2014), so it is unlikely to regulate the catalytic activity of PL2 pro . In agreement with this conclusion, the presence or absence of the Ubl2 of SARS-CoV or MERS-CoV shows almost no effect on the PL2 pro activities (Frieman et al, 2009;Clasman et al, 2017).…”

“…The PL2 pro adopts an extended right-hand fold with thumb, palm, and fingers subdomains, similar to the TGEV PL1 pro (Fig. 6; Ratia et al, 2006;Lei et al, 2014;Lee et al, 2015;Kong et al, 2015;Chen et al, 2015;Clasman et al, 2017; see also Table 1) and human USPs (e.g. USP14, USP7; Ratia et al, 2006).…”

“…In contrast, Clementz et al (2010) reported that the Ubl2 of SARS-CoV is not necessary for antagonizing IFN production. Also, Mielech et al (2015) showed that the Val787Ser mutation (Nsp3 numbering) in the MHV Ubl2 reduces the thermal stability of the PL2 pro , whereas Clasman et al (2017) reported that the deletion of Ubl2 in MERS-CoV did not affect PL2 pro thermal stability. The former Val residue of MHV is conserved in SARS-CoV and MERS-CoV.…”

“…Furthermore, the authors also reported that this inhibitor shows very high selectivity against human ubiquitin C-terminal hydrolase (hUCH-L1; IC 50 > 100 μM), which is one of the host proteins most closely related to the CoV PL pro (Lee et al, 2015). In contrast, Clasman et al (2017) reported that compound 4 features no selective inhibition of CoV PL pro s nor host USPs; therefore, this compound could be a pan-assay interference inhibitor (or PAIN).…”

Porcine epidemic diarrhea virus papain-like protease 2 can be noncompetitively inhibited by 6-thioguanine

“…S1) and homology modeling, PEDV PL2 pro may be composed of four domains: ubiquitin-like (Ubl), palm, thumb and fingers domains. The latter three domains form a catalytic core and may retain catalytic activity, like that of SARS-and MERS-CoV PL pro s (Chou et al, 2012;Clasman et al, 2017;Lei et al, 2014). In the present study, we made a recombinant catalytic core PEDV PL2 pro and demonstrated its secondary, tertiary and quaternary structural characteristics.…”