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

FEBS Letters

Maeda, Tatsuya²  Hata, Shoji¹  Abe, Keiko¹  Sorimachi, Hiroyuki¹  Nakagawa, Kazuhiro¹  Suzuki, Koichi³ 

[1] Laboratory of Biological Function, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan;Laboratory of Molecular Structure and Function, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan;Tokyo Metropolitan Institute of Gerontology, 35-2, Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan

关键词: Calpain;    Calpain activation;    Ca2+ binding;    Protease domain;    Cysteine protease;    EF-hand motif;   

DOI  :  10.1016/S0014-5793(01)02611-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Calpain, a Ca²⁺-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca²⁺-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca²⁺, 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 Ca²⁺-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 Ca²⁺-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 Ca²⁺-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca²⁺-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 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 complete activation of calpain.

【 授权许可】

Unknown