A Human RNA Viral Cysteine Proteinase That Depends upon a Unique Zn(2+)-binding Finger Connecting the Two Domains of a Papain-like Fold

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 Gly(111) and Asn(112), far upstream of its own catalytic residue Cys(1054). In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn(2+) finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn(2+) 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 Zn(2+), which could be partly substituted by Co(2+) during renaturation. The reconstituted, Zn(2+)-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn(2+)-depleted protein was not reactivated by adding Zn(2+) after renaturation. Consistent with the proposed essential structural role of Zn(2+), PL1pro was selectively inactivated by mutations in the Zn(2+) finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn(2+). 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.