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

钙蛋白酶由80K和30K两个亚基组成，但80K本身表达完整的酶活性。对无钙条件下钙蛋白酶的X-射线分析结果表明，80K的蛋白水解区(II)由两个亚区(II和IIB)组成，它们与其他半胱氨酸蛋白酶中的亚区非常相似。然而，它们是分开的，并且没有形成存在于两个子域之间的适当的活性部位。通过钙诱导的构象变化，研究了这两个亚区如何更接近形成合适的活性中心的激活机制。这两个亚区主要通过两种相互作用来分隔，即30K时I与Cahnode in结构域(VI)的相互作用和Lib与III的相互作用。当Ca与VI和III结合时，这些相互作用被破坏，导致较大的构象变化，使两个亚结构域更接近。此外，钙与II结合引起的微小构象变化是使II活性的关键。IL1在这种激活中起着非常重要的作用，它既是钙结合的新位点，也是决定活性钙蛋白细胞定位的磷脂结合域。在II中还预测了一个新的钙结合部位，但其确切位置仍不清楚。了解钙结合形式的钙蛋白酶的结构是理解最终激活机制的关键。沿着这条线，进一步的实验现在正在进行中。

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.