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

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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 β‐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 α‐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.