| Family type peptidase | C18.001 - hepatitis C virus peptidase 2 (hepatitis C virus), MEROPS Accession MER0000812 (peptidase unit: 810-1026) |
| Content of family | Peptidase family C18 contains a viral cysteine endopeptidase. |
| History |
Identifier created: Methods Enzymol. 244:461-486 (1994)
The most studied endopeptidase of the hepatitis C virus (HCV) is hepacivirin (S29.001), also known as the NS3 protease. Mutation of the active-site nucleophile of hepacivirin, Ser1165, abolished cleavage at four downstream sites in the polyprotein, but had little or no effect on cleavage at the NS2/NS3 site (Leu1026 Ala1027), indicating that a distinct peptidase is responsible for cleavage at this "2/3" site (Bartenschlager et al., 1993; Grakoui et al., 1993; Hijikata et al., 1993). The discovery that approximately 130 amino acids upstream and 180 downstream of the 2/3 site were required for cleavage, coupled with the observation that certain mutations in NS2 prevented or inhibited 2/3 cleavage, suggested that cleavage at this site is catalyzed by a virus-encoded proteolytic activity residing in the NS2–NS3 region of the polyprotein. |
| Catalytic type | Cysteine |
| Active site residues | H952 E972 C993 |
| Active site | From the crystal structure, there is an active site triad which occurs in the order His, Glu, Cys in the sequence (Lorenz et al., 2006). Mutations His952Ala or Cys993Ala eliminated activity of hepatitis C virus endopeptidase 2 leaving that of hepacivirin unaffected (Grakoui et al., 1993; Hijikata et al., 1993). In the same studies some other mutations affected the 2/3 cleavage though less selectively; these included Cys1123Ala, Cys1125Ala and Cys1171Ala. These latter three cysteine residues together with a histidine form a tetradentate (presumably structural) zinc-binding site (De Francesco et al., 1996; Love et al., 1995). |
| Activities and specificities | Cleavage of the 2/3 site is thought to occur in cis (i.e. intramolecularly), partly because it is very rapid (Marcotrigiano et al., 2004). Activity is enhanced by microsomal membranes and certain detergents (Pieroni et al., 1997). |
| Inhibitors | Activity is decreased by EDTA, and increased by zinc ions (Hijikata et al., 1993). No cysteine residue in the endopeptidase sequence could be identified as highly-reactive (Orsatti et al., 2002). The cellular chaperone, HSP90, was identified as an essential factor for endopeptidase 2, and inhibitors of the ATPase activity of the chaperone were inhibitory for the peptidase (Waxman et al., 2001. |
| Molecular structure | The tertiary structure of the catalytic domain has been determined and has been shown to be a unique fold (Lorenz et al., 2006). The active peptidase is a homodimer, and each monomer comprises an N-terminal, helical subdomain, a C-terminal subdomain with a beta sheet and an extended linker region. The active site His and Glu are situated in the N-terminal domain and the Cys in the C-terminal domain. An active site is only formed in the dimer, when the His and Glu from one monomer oppose the Cys from the other. The dimer has two active sites. Because the C-terminal residues remain co-ordinated in the active sites, the post-cleavage form of the peptidase is probably inactive. The structural zinc-binding site is C-terminal to the domain in the crystallized form. |
| Clan | CM |
