Domain II of m-calpain is a Ca2+-dependent cysteine protease

Calpain, a Ca2+-dependent cytosolic cysteine protease, proteolytically modu lates specific substrates involved in Ca2+-mediated intracellular events, s uch as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m- calpain, in the absence of Ca, revealed that the two su bdomains (domains IIa and IIb) of the protease domain (II) have an 'open' c onformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca2+-binding structure was identified in the 3D structure. The refore, it is predicted that if the protease domain is excised from the cal pain molecule, it will have a Ca2+-independent protease activity. In this s tudy, we have characterized a truncated human m-calpain that consists of on ly the protease domain. Unexpectedly, the proteolytic activity was Ca2+-dep endent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca2+-dependent modification of the protease domain by the cystei ne protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a res ult of Ca2+ binding. These results suggest that the Ca2+ binding to domain II, as well as to domains III, IV, and VI, is critical in the process of co mplete activation of calpain. (C) 2001 Federation of European Biochemical S ocieties. Published by Elsevier Science BY. All rights reserved.