Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C2-like domain

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca2+-dependent cysteine proteases that require surprisingly high Ca2+ concentrations for activation in vitro (similar to 50 and similar to 300 muM, respectively), The molecular basis of such a high requirement for Ca2+ in vitro is not known. Tn 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 C-2-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 mu- 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 EL-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 TV 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.