Abstract
A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with theEscherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.
Highlights
From the ‡Institute of Virology and Immunology, University of Wurzburg, Versbacher Strasse 7, 97078 Wurzburg, Germany, the ¶Advanced Biomedical Computing Center, SAIC/NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702-1201, ʈLeiden University Medical Center, AZL P4 –22, P.O
Optical emission spectrometry has been used to confirm the presence of Zn2؉ in a purified and proteolytically active form of the human coronavirus 229E (HCoV) PL1pro fused with the Escherichia coli maltose-binding protein
Proteolytic enzymes employing different catalytic mechanisms within the framework of the same structural fold have been identified (showing that protease families can extend across the borders separating classes (4 – 6)) and proteases that are decorated with determinants of nonproteolytic activity, for example, polynucleotidebinding cysteine proteases [7,8,9] and Zn2ϩ-binding nonmetalloproteases have been described
Summary
Computer Sequence and Structure Analyses—Amino acid sequences were derived from Swiss-Prot and GenbankTM data bases, and protein structures were derived from the Brookhaven data base (Protein Data Bank, Brookhaven National Laboratory, Upton, NY). Nonredundant sequence data bases were searched with single sequences [46], blocks [47], and Hidden Markov Models trained on multiple sequence alignments [48] These alignments were sent as input for the PhD [49] or DSC [50] programs to predict secondary structure. In Vitro Trans-cleavage Assay—The proteolytic activity of in vitro synthesized proteins expressed from pT7-IRES-Pap and its derivatives or the activity of bacterially expressed MBP-PL1 was assayed using in vitro synthesized, [35S]Met-labeled substrate, representing aa 1–956 of pp1a/pp1ab [41]. The aliquots were subsequently dialyzed twice against 500 volumes of buffer A containing 1 mM EDTA (aliquot I) or 100 M ZnOAc (aliquot II) at 4 °C for 16 h. All samples were treated twice with Chelex-100 resin by mixing in suspension at 4 °C for 30 min
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