Hosfield C M, Elce J S, Davies P L, Jia Z
Department of Biochemistry, Queen's University and The Protein Engineering Network of Centres of Excellence, Kingston, Ontario, Canada K7L 3N6.
EMBO J. 1999 Dec 15;18(24):6880-9. doi: 10.1093/emboj/18.24.6880.
The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.
硫醇蛋白酶活性与类钙调蛋白EF手结构域的结合是钙蛋白酶特有的特征。调控钙蛋白酶活性的机制很复杂，在缺乏结构信息的情况下，Ca(2+)诱导的无活性形式与活性形式之间转换的本质仍不清楚。我们在此描述了无Ca(2+)形式的m-钙蛋白酶的2.6埃晶体结构，该结构阐明了在没有Ca(2+)时钙蛋白酶无活性的结构基础。它还揭示了一种不寻常的硫醇蛋白酶折叠结构，该结构通过异源二聚化和一个类似C(2)的β-折叠结构域与Ca(2+)结合结构域相关联。引人注目的是，该结构表明催化三联体未组装，这表明Ca(2+)结合必须诱导构象变化，从而使蛋白酶结构域重新定向以形成功能性活性位点。催化亚基的α-螺旋N端锚定结构不占据活性位点，但会抑制其组装，并通过与调节亚基结合来调节Ca(2+)敏感性。这种Ca(2+)依赖性激活机制与经典蛋白酶的机制明显不同。
