Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease

Abstract

Papain-like protease (PLpro) from severe acute respiratory syndrome (SARS) coronavirus is one of the two proteases involved in the proteolytic processing of the virion polyproteins. In addition, PLpro shows significant in vitro deubiquitinating and de-ISGylating activities. All these findings demonstrated the multifunctional nature of the PLpro. Here we report the sensitivity of PLpro to denaturant urea. An increase in urea concentration induced a reversible biphasic unfolding of the enzyme. Differently, the unfolding of the catalytic triad region located within the palm and thumb domains followed a monophasic unfolding curve. Further observations suggest that the zinc-binding domain may start to unfold during the first transition. An 80% lost of its enzymatic activity at a urea concentration lower than 1M showed a close correlation with unfolding of the zinc-binding domain. The enzyme was also characterized in terms of hydrophobicity and size-and-shape distribution. We have demonstrated that PLpro displayed differential domain structure stability and molten globule state in its folding. These studies will not only assist in our understanding of the folding of this viral enzyme, but also that of other deubiquitinating enzymes with a similar scaffold.

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes

“…Not surprisingly, the melting temperature of both PL pro s decreased 10-15°C in the presence of disulfiram. These results conform to our earlier finding that the release of zinc ion can destabilize PL pro (Chou et al, 2012). 7)).…”

“…5A, left panel). Previous studies have suggested that the release of zinc ion following treatment with EDTA will lead to a 62% loss of PL pro activity (Chou et al, 2012). This result is consistent with the effect of disulfiram on PL pro s. Also, the inability of the reductant to rescue the DUB activity of MERS-CoV PL pro , suggesting that disulfiram cannot influence its active site, is compatible with disulfiram's noncompetitive mode of inhibition of the enzyme.…”

“…The forward primer was 5′-gtacactggtaactatcaggcgggtcatt acactcatata and the reverse primer was 5′-tatatgagtgtaatgacccgcctgatagttaccagtgtac. The MERS-CoV and SARS-CoV PL pro s and the SARS-CoV PL pro C271A mutant protein were produced and purified as previously described (Chou et al, 2012Lin et al, 2014). Briefly, the cultures were grown at 37°C for 4 h, then induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and grown at 20°C for 20 h. The cell pellet was resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, 2 mM β-mercaptoethanol (βME)), lysed by sonication and then centrifuged to remove the insoluble pellet.…”

Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus

“…The protein induction and purification procedures for SARS-CoV, the E168R mutant of SARS-CoV PL pro and MERS-CoV PL pro have been described previously (Chou et al, 2012Lin et al, 2014). The expression plasmid for MERS-CoV PL pro (GenBank accession number NC_019843.2; polyprotein residues 1484-1800) includes 6Â His tag at the C-terminus (Lin et al, 2014).…”

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.…”