Origins of the difference in Ca2+ requirement for activation of μ- and m-calpain

The mu- and m-calpains are closely related Ca2+-dependent cysteine proteases having different in vitro Ca2+ requirements (K-d), of approx. 25 and 325 muM 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 Ca2+ required for the first phase of calpain activation and do not reflect the lower Ca2+ 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 Ca2+ 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.