The physiological role of the skeletal muscle-specific calpain 3, p94, is presently unknown, but defects in its gene cause limb girdle muscular dystrophy type 2A. This calcium-dependent cysteine protease resembles the large subunit of m-calpain but with three unique additional sequences: an N-terminal region (NS), and two insertions (IS1 and IS2). The latter two insertions have been linked to the chronic instability of the whole enzyme both in vivo and in vitro. We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a recombinant protein in the absence of Ca 2؉ and undergoes autolysis in its presence. Here we show that p94I-II cannot hydrolyze an exogenous substrate before autolysis but is increasingly able to do so when autolysis proceeds for several hours. This gain in activity is caused by cleavage of IS1 during autolysis because a deletion mutant lacking the NS region (p94I-II ⌬NS) shows the same activation profile. Similarly, the calpain inhibitors E-64 and leupeptin have almost no inhibitory effect on substrate hydrolysis by p94I-II soon after calcium addition but cause complete inhibition when autolysis progresses for several hours. As autolysis proceeds, there is release of the internal IS1 peptide, but the two portions of the core remain tightly associated. Modeling of p94I-II suggests that IS1 contains an amphipathic ␣-helix flanked by extended loops. The latter are the targets of autolysis and limited digestion by exogenous proteases. The presence and location of the ␣-helix in recombinant IS1 were confirmed by circular dichroism and by the introduction of a L286P helix-disrupting mutation. Within p94I-II, L286P caused premature autoproteolysis of the enzyme. IS1 is an elaboration of a loop in domain II near the active site, and it acts as an internal autoinhibitory propeptide, blocking the active site of p94 from substrates and inhibitors.Calpains comprise a large family of cytosolic Ca 2ϩ -dependent cysteine proteinases with homologues present in mammals, insects, nematodes, and yeast (1, 2). Two well-studied members of this family are the mammalian heterodimeric -and mcalpains, which are composed of a large catalytic subunit (80 kDa) and a small regulatory subunit (28 kDa). The domain structure of the large subunit was redefined by x-ray crystallography (3, 4). Following the classification of Hosfield et al. (3), the large subunit has four structural domains (I-IV). The first two (I and II) make up the papain-like catalytic core common to all calpains (1), whereas domains III and IV are the C2-like and penta-EF-hand domains, respectively. The small subunit contains two domains, V and VI, of which the latter is also a penta-EF-hand domain that forms extensive interactions with the homologous penta-EF-hand domain IV in the large subunit. Several more of the ϳ14 human calpain isoforms are composed of two subunits and share the same domain structure as -and m-calpain. Other calpain homologues do not seem to form heterodimers, and some also contain different ...

Calpains 1 and 2 are heterodimeric proteases in which large (relative molecular mass M(r) 80000) and small (M(r) 28000) subunits are linked through their respective PEF (penta-EF-hand) domains. The skeletal muscle-specific calpain 3 is believed not to form a heterodimer with the small subunit but might homodimerize through its PEF domain. Size-exclusion chromatography and analytical ultracentrifugation of the recombinant PEF domain of calpain 3 show that it forms a stable homodimer that does not dissociate on dilution. Molecular modelling suggests that there would be no barriers to the dimerization of the whole enzyme through the PEF domains. This orientation would place the catalytic centres at opposite ends of the dimer.

pose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold. Key words: Calpastatin / Kininogen / Papain / Stefin B / Surface plasmon resonance/ Temporary inhibition. IntroductionThe two ubiquitous calpains, µ-calpain and m-calpain, are intracellular, Ca 2+ -dependent cysteine proteinases that have been implicated in many important cellular functions and various pathologies (see Sorimachi et al., 1997;Carafoli and Molinari, 1998;Suzuki and Sorimachi, 1998 for recent reviews). They consist of distinct (yet homologous) large (L-)subunits (80 kDa) und a common small (S-)subunit (30 kDa). On the basis of sequence comparisons, the L-subunit has been predicted to contain four and the S-subunit two domains. Whereas the catalytic domain (domain II) of the L-subunit shows a weak sequence homology to papain, both the L-subunit and the S-subunit contain a Ca 2+ -binding calmodulin-like domain (CaMLD). Until recently, only three-dimensional structures of the L-CaMLD were known (Blanchard et al., 1997;Lin et al ., 1997), and hypotheses on the molecular mechanisms of activation and inhibition of calpains have been contradictory. Meanwhile, two groups have published crystal structures of the Ca 2+ -free, inactive form of m-calpain, revealing the molecular architecture of this multidomain protein (Hosfield et al., 1999;Strobl et al., 2000). In these structures, the catalytic domain (II) appears disrupted into two subdomains (IIa and IIb), explaining the inactivity of calpain in the absence of calcium. Activation should involve a 'fusion' of the two subdomains, leading to a functional papain-like catalytic domain (Hosfield et al., 1999;Strobl et al., 2000). As long as the structure of a Ca 2+ -activated calpain is not known, a number of questions concerning the molecular mechanisms of interaction with substrates and inhibitors remain open.Ca 2+ -activated µ-and m-calpain are controlled by a very specific intracellular protein inhibitor, calpastatin. Calpastatin contains four repeats of the inhibitory unit, each of which can inhibit calpain independently, but is not able to inhibit other cysteine proteinases of the papain superfamily (Maki et al ., 1987;Emori et al ., 1988 Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit -and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and ⌬L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefinB by the corresponding KD2 sequence resulted in a calpain inhibitor of K i = 188 nM. Deletion of L110, which forms a ␤-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of -calpain 4-to 8-fold. All engineer...

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