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

基于钙蛋白酶三级结构的激活机制分析

基本信息

批准号：
12308032
负责人：
SUZUKI Koichi
金额：
$ 21.7万
依托单位：
Tokyo Metropolitan Institute of Gerontology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12308032/
关键词：
Calpain Calcium Protease X ray structural analysis Higher order structure of protein Activation mechanism Phopsholipids 蛋白質の構造変化リン脂質

项目摘要

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.

钙蛋白酶由两个亚基组成，分别为80k和30k，但80k本身表达了完全的酶活性。在不存在CA的情况下，Calpain的X射线分析的结果表明，包含结构域（I-IV）的80K的蛋白酶结构域（II）由两个子域II和IIB组成，II和IIB与其他半胱氨酸蛋白酶中的蛋白酶（I-IV）非常相似。但是，它们是分开的，并且没有形成两个子域之间存在的适当的活跃位点。研究了与这两个子域如何通过CA引起的构象变化更接近适当的活动位置的激活机制。两个子域主要通过两种相互作用分离，即30K中的I和Cahnodulin域（VI）之间的相互作用，以及LIB与III的相互作用。在与VI和III结合时，这些相互作用被破坏，导致构象变化，使两个子域变得更加接近。此外，CA与II结合引起的次要构象变化对于使II活跃至关重要。生病在这种激活中扮演着非常重要的角色。充当新型的Ca结合位点，也充当决定活性钙蛋白酶细胞定位的phopspholipids结合结构域。在II中还预测了一个新型的Ca结合位点，但其精确位置仍然未知。要了解CA-BON形式的CALPAIN的结构对于了解最终激活机制至关重要。现在，进一步的实验正在沿着这条线进行。