Dissociation of m-Calpain Subunits Occurs after Autolysis of the N-Terminus of the Catalytic Subunit, and Is Not Required for Activation

Calpain is a heterodimeric, intracellular Ca2+-dependent, “bio-modulator” that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-tenninus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into sub-units is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca2+ has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (ç19), but not those lacking only nine residues (ç9), dissociated into subunits even in the absence of Ca2+. Moreover, both ç9 and ç19 mutants showed an equivalent reduced Ca2+ requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.