Abstract

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 Å 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 C₂-like -sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca²⁺-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The -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.