Pfam PF01707: Peptidase family C9 (family)
The family of alphaviruses includes 26 known members. They infect a variety of hosts including mosquitoes, birds, rodents and other mammals with worldwide distribution. Alphaviruses also pose a potential threat to human health in many area. For example, Venezuelan Equine Encephalitis Virus (VEEV) causes encephalitis in humans as well as livestock in Central and South America, and some variants of Sinbis Virus (SIN) and Semliki Forest Virus (SFV) have been found to cause fever and arthritis in humans .

Alphaviruses possess a single-stranded RNA genome of approximately 12 kb. The genomic RNA of alphaviruses is translated into two polyproteins that, respectively, encode structural proteins and nonstructural proteins. The nonstructural proteins may be translated as one or two polyproteins, nsp123 or nsp1234, depending on the virus. These polyproteins are cleaved to generate nsp1, nsp2, nsp3 and nsp4 by a protease activity that resides within nsp2. The nsp2 protein of alphaviruses has multiple enzymatic acivities. Its N-terminal domain has been shown to possess ATPase and GTPase activity, RNA helicase activity and RNA 5'-triphosphatase activity. The C-terminal nsp2 protease (nsp2pro) domain of nsp2 is responsible for the regulation of 26S subgenome RNA synthesis, switching between negative- and positive-strand RNA synthesis, targeting nsp2 for nuclear transport and proteolytic processing of the nonstructural polyprotein . The nsp2pro domain is a member of peptidase family C9 of clan CA.

The nsp2pro domain consists of two distinct subdomains. The nsp2pro N-terminal subdomain is largely Î±-helical and contains the catalytic dyad cysteine and histidine residues organised in a protein fold that differs significantly from any known cysteine protease or protein folds. The nsp2pro C-terminal subdomain displays structural similarity to S-adenosyl- L-methionine-dependent RNA methyltransferases and provides essential elements that contribute to substrate recognition and may also regulate the structure of the substrate binding cleft .

This domain covers the entire nsp2pro domain.
A cysteine peptidase is a proteolytic enzyme that hydrolyses a peptide bond using the thiol group of a cysteine residue as a nucleophile. Hydrolysis involves usually a catalytic triad consisting of the thiol group of the cysteine, the imidazolium ring of a histidine, and a third residue, usually asparagine or aspartic acid, to orientate and activate the imidazolium ring. In only one family of cysteine peptidases, is the role of the general base assigned to a residue other than a histidine: in peptidases from family C89 (acid ceramidase) an arginine is the general base. Cysteine peptidases can be grouped into fourteen different clans, with members of each clan possessing a tertiary fold unique to the clan. Four clans of cysteine peptidases share structural similarities with serine and threonine peptidases and asparagine lyases. From sequence similarities, cysteine peptidases can be clustered into over 80 different families . Clans CF, CM, CN, CO, CP and PD contain only one family.

Cysteine peptidases are often active at acidic pH and are therefore confined to acidic environments, such as the animal lysosome or plant vacuole. Cysteine peptidases can be endopeptidases, aminopeptidases, carboxypeptidases, dipeptidyl-peptidases or omega-peptidases. They are inhibited by thiol chelators such as iodoacetate, iodoacetic acid,N-ethylmaleimide orp-chloromercuribenzoate.

Clan CA includes proteins with a papain-like fold. There is a catalytic triad which occurs in the order: Cys/His/Asn (or Asp). A fourth residue, usually Gln, is important for stabilising the acyl intermediate that forms during catalysis, and this precedes the active site Cys. The fold consists of two subdomains with the active site between them. One subdomain consists of a bundle of helices, with the catalytic Cys at the end of one of them, and the other subdomain is a Î²-barrel with the active site His and Asn (or Asp). There are over thirty families in the clan, and tertiary structures have been solved for members of most of these. Peptidases in clan CA are usually sensitive to the small molecule inhibitor E64, which is ineffective against peptidases from other clans of cysteine peptidases .

Clan CD includes proteins with a caspase-like fold. Proteins in the clan have an Î±/Î²/Î± sandwich structure. There is a catalytic dyad which occurs in the order His/Cys. The active site His occurs in a His-Gly motif and the active site Cys occurs in an Ala-Cys motif; both motifs are preceded by a block of hydrophobic residues . Specificity is predominantly directed towards residues that occupy the S1 binding pocket, so that caspases cleave aspartyl bonds, legumains cleave asparaginyl bonds, and gingipains cleave lysyl or arginyl bonds.

Clan CE includes proteins with an adenain-like fold. The fold consists of two subdomains with the active site between them. One domain is a bundle of helices, and the other a Î²-barrel. The subdomains are in the opposite order to those found in peptidases from clan CA, and this is reflected in the order of active site residues: His/Asn/Gln/Cys. This has prompted speculation that proteins in clans CA and CE are related, and that members of one clan are derived from a circular permutation of the structure of the other.

Clan CL includes proteins with a sortase B-like fold. Peptidases in the clan hydrolyse and transfer bacterial cell wall peptides. The fold shows a closed Î²-barrel decorated with helices with the active site at one end of the barrel . The active site consists of a His/Cys catalytic dyad.
Cysteine peptidases with a chymotrypsin-like fold are included in clan PA, which also includes serine peptidases. Cysteine peptidases that are N-terminal nucleophile hydrolases are included in clan PB. Cysteine peptidases with a tertiary structure similar to that of the serine-type aspartyl dipeptidase are included in clan PC. Cysteine peptidases with an intein-like fold are included in clan PD, which also includes asparagine lyases.