logo CaMPDB: Calpain for Modulatory Proteolysis Database

Calpains (EC, Clan CA, family C02) in mammals co-exist in cells with the very specific endogenous inhibitor protein, “calpastatin”, strongly suggesting the pivotal role of this inhibitor in the regulation of calpain activity (Goll et al. 1992; Kawasaki & Kawashima 1996; Maki et al. 1991; Menard & el-Amine 1996; Nixon et al. 1994). Calpastatin was first cloned in 1987 (Emori et al. 1987) and then found not to have exact secondary structures (Uemori et al. 1990). Calpastatin contains four equivalent inhibitory domains of ca. 140 residues having three conserved regions, A, B, and C, important for inhibition (Fig. 1). A and C interact with IV and VI, respectively, in a Ca2+-dependent manner, and B shows inhibitory activity by itself probably by binding at or near the active site (Tompa et al. 2002) (Fig. 2). Presence of two calmodulin-like domains IV and VI are necessary for effective inhibition by calpastatin. Thus calpastatin inhibits only dimeric calpain, namely μ-calpain, m-calpain, and nCL-4/CAPN9 with 30K. Calpain large subunit homologues including p94/CAPN3 and nCL-2/CAPN8 are not inhibited and escape from the regulation by calpastatin (Hata et al. 2001; Ono et al. 2004; Sorimachi et al. 1993b).


Fig. 1. Schematic structure of human calpastatin Calpastatin has four repetitive inhibitory domains (1~4), which can inhibit one molecule of heterodimer calpains, although their inhibitory activities vary. At the N-terminus, there are two extended domains, XL and L, whose functions are unknown. Each inhibitory domain has three regions, A, B, and C, which binds to domain IV of catalytic subunit, the active site of domain II, and domain VI of regulatory subunit (see Fig. 2), respectively. Peptide corresponding to only the Region B has inhibitor activity, which is less than full-length domain. The Region B has highly conserved sequences at the center, whose consensus is GxxE/DxTIPPxYR.


Fig. 2. Three-dimensional structure of 30K homodimer biding to calpastatin region C and synthetic calpain inhibitor molecule PD150606 A peptide corresponding to the Region B (see Fig. 1) of human calpastatin binds to the first α-helix (E-helix) of the first EF-hand motif (EF-1) of both 30K molecule. PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid), which is a unique synthetic inhibitor molecule for calpains not directing to the active site, binds close to the loop region of EF-3 and E-helix of EF-5. Cross-eye representation.

All sections ≫ open : close

1. Structure of calpastatin

2. Other synthetic inhibitors of calpain

3. References