1. Department of Immunology, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, Tennessee 38105, USA
2. Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada

Correspondence to: Douglas R. Green¹ Correspondence and requests for materials should be addressed to D.R.G. (Email: douglas.green@stjude.org).

Abstract

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.

1. Department of Immunology, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, Tennessee 38105, USA
2. Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada

Correspondence to: Douglas R. Green¹ Correspondence and requests for materials should be addressed to D.R.G. (Email: douglas.green@stjude.org).

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