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The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx. 25 and 325 microM respectively. The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits. As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d). In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90%. K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding. Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.