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 (K-d). For m-calpain, K-d is similar to325 muM, for mu-calpain it is similar to50 muM, and for calpain 3 it is not strictly known but may be similar to0.1 muM. 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 K-d. The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K-d 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 beta-sheet interaction involving Glu517 are factors contributing to the high K-d 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 L-calpain and in calpain 3 are also consistent with their progressively lower K-d values. (C) 2004 Elsevier Ltd. All rights reserved.