Peptidase C2, calpain, catalytic domain (IPR001300)
Short name: Peptidase_C2_calpain_cat
This group of cysteine peptidases belong to the MEROPS peptidase family C2 (calpain family, clan CA). A type example is calpain, which is an intracellular protease involved in many important cellular functions that are regulated by calcium [PMID: 2539381,PMID: 11517928]. The protein is a complex of 2 polypeptide chains (light and heavy), with eleven known active peptidases in humans and two non-peptidase homologues known as calpamodulin and androglobin [PMID: 21864727]. These include a highly calcium-sensitive (i.e., micro-molar range) form known as mu-calpain, mu-CANP or calpain I; a form sensitive to calcium in the milli-molar range, known as m-calpain, m-CANP or calpain II; and a third form, known as p94, which is found in skeletal muscle only [PMID: 2555341].
All forms have identical light but different heavy chains. Both mu- and m-calpain are heterodimers containing an identical 28kDa subunit and an 80kDa subunit that shares 55-65% sequence homology between the two proteases [PMID: 7845226, PMID: 2539381]. The crystallographic structure of m-calpain reveals six "domains" in the 80kDa subunit [PMID: 9396712,PMID: 11328585]:
- A 19-amino acid NH2-terminal sequence;
- Active site domain IIa;
- Active site domain IIb. Domain 2 shows low levels of sequence similarity to papain; although the catalytic His has not been located by biochemical means, it is likely that calpain and papain are related [PMID: 7845226].
- Domain III;
- An 18-amino acid extended sequence linking domain III to domain IV;
- Domain IV, which resembles the penta EF-hand family of polypeptides, binds calcium and regulates activity [PMID: 7845226]. Ca2+-binding causes a rearrangement of the protein backbone, the net effect of which is that a Trp side chain, which acts as a wedge between catalytic domains IIa and IIb in the apo state, moves away from the active site cleft allowing for the proper formation of the catalytic triad [PMID: 11914728].
Calpain-like mRNAs have been identified in other organisms including bacteria, but the molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in these organisms cells is still unclear In metazoans, the activity of calpain is controlled by a single proteinase inhibitor, calpastatin (IPR001259). The calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. The calpains ostensibly participate in a variety of cellular processes including remodelling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma [PMID: 12843408].
Calpains are a family of cytosolic cysteine proteinases (see PDOC00126). Members of the calpain family are believed to function in various biological processes, including integrin-mediated cell migration, cytoskeletal remodeling, cell differentiation and apoptosis [PMID: 11854009, PMID: 11950589].
The calpain family includes numerous members from C. elegans to mammals and with homologues in yeast and bacteria. The best characterised members are the m- and mu-calpains, both proteins are heterodimer composed of a large catalytic subunit and a small regulatory subunit. The large subunit comprises four domains (dI-dIV) while the small subunit has two domains (dV-dVI). Domain dI is a short region cleaved by autolysis, dII is the catalytic core, dIII is a C2-like domain, dIV consists of five calcium binding EF-hand motifs [PMID: 11950589].
The crystal structure of calpain has been solved [PMID: 10601010, PMID: 11893336]. The catalytic region consists of two distinct structural domains (dIIa and dIIb). dIIa contains a central helix flanked on three faces by a cluster of alpha-helices and is entirely unrelated to the corresponding domain in the typical thiol proteinases. The fold of dIIb is similar to the corresponding domain in other cysteine proteinases and contains two three-stranded anti-parallel beta-sheets. The catalytic triad residues (C,H,N) are located in dIIa and dIIb. The activation of the domain is dependent on the binding of two calcium atoms in two non EF-hand calcium binding sites located in the catalytic core, one close to the Cys active site in dIIa and one at the end of dIIb. Calcium-binding induced conformational changes in the catalytic domain which align the active site [PMID: 11893336][PMID: 11914728].
The profile covers the whole catalytic 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 [PMID: 11517925]. 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 or p-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 beta-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 [PMID: 7044372].
Clan CD includes proteins with a caspase-like fold. Proteins in the clan have an alpha/beta/alpha 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 [PMID: 9891971]. 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 beta barrell. 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 beta barrel decorated with helices with the active site at one end of the barrel [PMID: 14725770]. 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.