Abstract
Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase‐7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase‐7 is allosterically inactivated when two of the substrate‐binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop‐locked version of caspase‐7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase‐7 structure in which the substrate‐binding groove is properly ordered even in the absence of an active‐site ligand. In the reactivated structure, the catalytic‐dyad cysteine–histidine are positioned 3.5 Å apart in an orientation that is capable of supporting catalysis. This redox‐controlled version of caspase‐7 is particularly well suited for targeted cell death in concert with redox‐triggered delivery vehicles.