Crystal structure of calpain reveals the structural basis for Ca2+-dependent protease activity and a novel mode of enzyme activation

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 Ca2+-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 Angstrom crystal structure of m-calpain in the Ca2+-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca2+. It also reveals an unusual thiol protease fold, which is associated with Ca2+-binding domains through heterodimerization and a C-2-like beta-sandwich domain. Strikingly, the structure shows that the catalytic Mad is not assembled, indicating that Ca2+-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 Ca2+-sensitivity through association with the regulatory subunit, This Ca2+-dependent activation mechanism is clearly distinct from those of classical proteases.