Cystatins as Calpain Inhibitors: Engineered Chicken Cystatin- and Stefin B-Kininogen Domain 2 Hybrids Support a Cystatin-Like Mode of Interaction with the Catalytic Subunit of μ-Calpain

摘要

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit μ- and m-calpain. In an attempt to elucidate the structural requirements of cystains for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and ΔL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of K_i = 188 nM. Deletion of L110, which forms a β-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of μ-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the μ-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.