Roles of individual EF-hands in the activation of m-calpain by calcium

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca²⁺-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca²⁺-binding EF-hands in each subunit, but their relative affinities for Ca²⁺ are not known. In the present study mutations were made in both subunits to reduce the Ca²⁺-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 Ca²⁺ 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 Ca²⁺ were otherwise identical to that of the Ca²⁺-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca²⁺ requirement (K(d)) was approx. 350 μM. 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 Ca²⁺ and provides the major driving force for conformational change.