Question

Primary sequence of Papain- like protease of Covid-19

Answer 1

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M−1s−1, 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

The identification of a coronavirus as the infectious agent of severe acute respiratory syndrome (SARS) and the rapid sequencing of its genome have led to a renewed interest in coronaviruses (34). SARS, which emerged in China in late 2002, is a pulmonary infection with a reported fatal outcome of 10% (42). The SARS coronavirus (SARS-CoV) is an enveloped, single-stranded positive-sense RNA virus with a genome comprising approximately 29,700 nucleotides. The SARS-CoV genome is predicted to encode a number of proteins, enzymes, and undefined domains (35, 46) whose functions, for the most part, are currently poorly characterized. The 5′ proximal region of the genome containing the replicase gene accounts for approximately two-thirds of the full-length genome and encodes the nonstructural proteins (nsp's) essential for viral replication as large overlapping replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). These polyproteins are processed by two viral cysteine proteases to release 16 nsp's (35, 46). The C-terminal regions of the pp1a and pp1ab polyproteins are processed at 7 and 11 sites, respectively, by a chymotrypsin-like cysteine protease (3CLpro, also called main protease). N-terminal processing of pp1a/pp1ab is carried out at three sites by a papain-like protease (PLpro), releasing nsp1, nsp2, and nsp3 .

The amino groups of sidechains of asparagine or glutamine residues in some PLPs could provide the stabilization forces required for the oxyanion hole. 14,29 The oxyanion hole contains a N99 in the corresponding position is found in SADS-CoV PLP2 structure. Besides N99, W102, which is conserved among coronavirus PLPs, is located over the catalytic cysteine, and may also contribute to the stabilization of oxyanion hole, because the side chain of W102 could form a hydrogen bond with an intermediate in the active site (Figure 2e). ...

... The catalytic cysteine (Cys101) is situated at the foot (N-terminus) of α-helix3 in the thumb domain, as the helix dipole could contribute to the microenvironment that stabilizes the thiolate of Cys during catalysis and this structural feature may also contribute to the nucleophilicity of Cys101. 14,29 The side chain sulfur atom of Cys101 is positioned 3.21 Å from the side-chain nitrogen atom of the catalytic-triad histidine (His256), which is located at the foot of the palm domain, and adjacent to the flexible loop BL2. One of the oxygen atoms of the side chain of catalytic aspartic acid (Asp269) is located 2.75 Å from the side-chain nitrogen of the catalytic histidine at the foot of the palm domain (Figure 2e). ...

... And the equivalent residues of SARS-CoV PLPro are W94 and D109, which formed the hydrogen bond essential for the catalytic activity. 23,29 Based on the consensus sequence of PLP2 for the three cleavage sites in the SADS-CoV polyprotein and biochemical studies addressing the substrate preference, it is proposed that a KXGG motif at the P4-P1 positions of the substrate is essential for the recognition and cleavage and also could explain why SADS-CoV PLP2 could cleave LRGG-AMC which is both the consensus sequences of SARS-CoV nsp1-4 cleavage sequence and the C-terminal sequences of ubiquitin and ISG15, even though with a lower k cat /K m , compared with KAGG-AMC. ...