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The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human mu-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive micro-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

active-site mutated heterodimeric human m-calpain with phospholipid bilayers was studied in vitro using proteinto-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca 2q -dependent, but not selective for specific phospholipid head groups. wCa 2q x 0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10-to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca 2q and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of m-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca 2q requirement for activation.

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...