Calpain Mutants with Increased Ca2+ Sensitivity and Implications for the Role of the C2-like Domain

The ubiquitous calpain isoforms (μ- and m-calpain) are Ca2+-dependent cysteine proteases that require surprisingly high Ca2+concentrations for activation in vitro (∼50 and ∼300 μm, respectively). The molecular basis of such a high requirement for Ca2+in vitro is not known. In this study, we substantially reduced the concentration of Ca2+ required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C2-like domain III were disrupted, and the effects of these mutations on activity and Ca2+sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both μ- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca2+requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca2+ requirement of calpain. The structures of the mutants in the absence of Ca2+ were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca2+ sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between μ-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca2+ binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II–domain III salt bridges are important in the process of the Ca2+-induced activation of calpain and that they influence the overall Ca2+ requirement of the enzyme.