Domain II of m-calpain is a Ca(2+)-dependent cysteine protease.
Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.
Hata
S
S
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
Sorimachi
H
H
Nakagawa
K
K
Maeda
T
T
Abe
K
K
Suzuki
K
K
D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002154:Calpain; D003546:Cysteine Endopeptidases; D002118:Calcium