Analysis of Activation Mechanism of Calpain on the Basis of its Tertiary Structure

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Calpain is composed of two subunits, 80K and 30K, but 80K itself expresses full enzyme activity. The results of X ray analysis of calpain in the absence of Ca, show that the protease domain (II) of 80K comprising domains (I-IV) is composed of two sub-domains, II and Iib, which are quite similar to those in other cysteine proteases. However, they are separated and the proper active-site that exists between the two sub-domains is not formed. The activation mechanism corresponding to how these two sub-domains come closer to forn proper active-site by conformational changes induced by Ca, was examined. Two sub-domains are separated mainly by two kinds of interactions, interaction between I and cahnodulin domain (VI) in 30K, and interaction of lib with III. Upon binding Ca to VI and III, these interactions are disrupted resulting in large conformational changes that make two sub-domains come closer. Further, minor conformational changes induced by Ca binding to II are essential to make II active. Ill plays very important roles in this activation ; acting as the novel Ca binding sites, and also as the phopspholipids binding domain that determines cellular localization of active calpain. A novel Ca binding site was also predicted in II but its precise location is still unknown. To know the structure of Ca-bound form of calpain is essential to understand the final activation mechanism. Further experiments are now in progress along this line.