Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-Å crystal structure of full-length heterodimeric [80-kDa (dI-dIV) ؉ 30-kDa (dV؉dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV؉dVI occupying opposite poles, and the tumor necrosis factor ␣-like ␤-sandwich domain dIII and the N-terminal segments dI؉dV located between. Compared with papain, the two subdomains dIIa؉dIIb of the catalytic unit are rotated against one another by 50°, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the Nterminal linkers.T he calpains (EC 3.4.22.17; Clan CA, family C02) are a family of calcium-dependent cytosolic cysteine proteinases. They seem to catalyze limited proteolysis of proteins involved in cytoskeletal remodeling and signal transduction but are also implicated in other physiological and pathophysiological processes, such as cell cycle regulation, apoptosis, muscular dystrophies, cataractogenesis, and Alzheimer's or Parkinson's diseases (1-5). In mammals, the calpain family comprises several ''tissuespecific'' isoforms (n-calpains) besides two ''ubiquitous'' isoenzymes (-and m-calpains). In lower organisms such as insects, nematodes, fungi, and yeast, a number of ''atypical'' calpain homologues have been found.The ubiquitous -and m-calpains (calpains I and II), by far the best characterized calpains, are heterodimers comprising distinct but quite homologous 80-kDa ''large'' L-subunits and a common 30-kDa ''small'' S-subunit. On the basis of amino acid homologies, the L-and S-subunits have been described as consisting of four domains, dI to dIV, and of two domains, dV and dVI, respectively, with domain dII somewhat resembling papain and the calmodulin-like domains dIV and dVI containing EF-hands (6, 7). On exposure to calcium at concentrations of 5-50 M (-calpain) and 200-1,000 M (m-calpain), both calpains are activated and partially autolyzed. In vivo, both calpains seem to be active at physiological calcium concentrations of 100-300 nM, however, suggesting that other factors such as phospholipids might play a role in activation in addition.The crystal structures of rat and porcine domain dVI in the absence and presence of calcium have been determined (8, 9). For a full understanding of the activation mechanism and the functioning of calp...

p94 (also called calpain 3) is the skeletal muscle-specific calpain and is considered to be a "modulator protease" in various cellular processes. Analysis of p94 at the protein level is an urgent issue because the loss of p94 protease activity causes limb-girdle muscular dystrophy type 2A. In this study, we enzymatically characterized one alternatively spliced variant of p94, p94:exons 6 ؊ 15 ؊ 16 ؊ (p94⌬), which lacks two of the p94-specific insertion sequences. In contrast to p94, which has hardly been studied enzymatically due to its rapid, thorough, and apparently Ca 2؉ -independent autolytic activity, p94⌬ was stably expressed in COS and insect cells. p94⌬ showed Ca 2؉ -dependent caseinolytic and autolytic activities and an inhibitor spectrum similar to those of the conventional calpains. However, calpastatin did not inhibit p94⌬ and is a substrate for p94⌬, which is consistent with the properties of p94, presenting p94 as a possible regulator of the conventional calpain system. We also established a semi-quantitative fluorescence resonance energy transfer assay using the calpastatin sequence specifically to measure p94 activity. This method detects the activity of COS-expressed p94 and p94⌬, suggesting that it has potential to evaluate p94 activity in vivo and in the diagnosis of limb-girdle muscular dystrophy type 2A.Calpain (EC 3.4.22.17, clan CA, family C2) is a Ca 2ϩ -requiring cysteine protease representing one of the most important families of the cysteine proteases (1-9). To date, various molecules showing significant similarity to the calpain protease domain have been identified in almost all kinds of living organisms and constitute the "calpain superfamily" (6). Two representative members, -and m-calpains, the so-called "conventional" calpains, are ubiquitously expressed and have been well characterized. These two calpains consist of a distinct larger catalytic subunit containing a protease domain (-or m-calpain large subunit, abbreviated as CL 1 or mCL, respectively) and a common smaller regulatory subunit (abbreviated as 30K according to its molecular weight). On the basis of amino acid similarities, the large and small subunits have been described as consisting of four and two domains, respectively, which agrees with the recently resolved three-dimensional structure of m-calpain (10, 11) (Fig. 1A).Conventional calpain has a specific endogenous proteinaceous inhibitor, calpastatin (12). Calpastatin contains four repetitive inhibitory units, each of which inhibits equimolar amounts of conventional calpain. The conserved reactive site interacts with the calpain protease domain, whereas the flanking ␣-helical regions bind to domains IV and VI of the large and small subunits, respectively. Synthetic oligopeptides (see Fig. 4D) corresponding to the calpastatin-reactive site specifically inhibit conventional calpain efficiently, although their inhibitory activity is weaker than that of the full-length inhibitory unit.The primary structure of p94 (also called calpain 3) is very similar to those of CL ...

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