Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains

Abstract

Calpains are cysteine proteases involved in remodelling protein structures needed for cell movement and division, in response to the release of calcium ions. They regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Two papers in this issue present a complete picture of how calpastatin shuts down calpain activity. Moldoveanu et al. determined the crystal structure of calcium-bound m-calpain in complex with the first calpastatin repeat, CID-1. Hanna et al. present the structure of the Ca2+-bound calpain 2 heterodimer bound to one of the other inhibitory domains of calpastatin, CID-IV. This study shows how the inhibitor binds and inhibits calpain only in the presence of calcium. More importantly, since calpastatin is itself a protein, we show the novel way in which calpastatin avoids being cut and destroyed by calpain. The 3.0 Å structure of Ca2+-bound m-calpain in complex with the first calpastatin repeat is solved, revealing the mechanism of the exclusive specificity. The structure highlights the complexity of calpain activation by Ca2+, illustrating key residues in a peripheral domain that serve to stabilize the protease core after Ca2+-binding. The Ca2+-dependent cysteine proteases, calpains, regulate cell migration1, cell death2, insulin secretion3, synaptic function4 and muscle homeostasis5. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency6. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown7. Here we report the 3.0 Å structure of Ca2+-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca2+, illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca2+ binding. Fully activated calpain binds ten Ca2+ atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.