Activation of Calpain by Ca2+: Roles of the Large Subunit N-terminal and Domain III–IV Linker Peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca2+ requirements (Kd). For m-calpain, Kd is ∼325 μM, for μ-calpain it is ∼50 μM, and for calpain 3 it is not strictly known but may be ∼0.1 μM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1–20) and a domain III–IV linker peptide (residues 514–530 in m-calpain) were important in defining Kd. The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced Kd of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca2+-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short β-sheet interaction involving Glu517 are factors contributing to the high Kd of m-calpain. Though these two regions are physically remote from the active site and Ca2+-binding site, they play significant roles in regulating the response of calpain to Ca2+. Differences in these interactions in μ-calpain and in calpain 3 are also consistent with their progressively lower Kd values.