Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of μ-calpain large subunit

Calpain requires Ca²⁺ both for proteolysis of its substrates and for interaction with its endogenous inhibitor, calpastatin. Although calmodulin-like domains (CaMLDs) of large and small subunits of calpain have been suggested to be the sites for Ca²⁺-dependent interaction with calpastatin, specificity and molecular basis of the interaction have remained unclear. We investigated the interaction between the CaMLD of human μ-calpain large subunit expressed in Escherichia coli and a 19-residue synthetic oligopeptide corresponding to the region A (the amino-terminal conserved acidic region) of one of the four repetitive functional domains of calpastatin. The recombinant CaMLD bound to the oligopeptide immobilized on Sepharose beads in a Ca²⁺-dependent manner. The CaMLD failed in binding to a mutant oligopeptide with one amino acid substitution. Enhancement of fluorescence intensity of a hydrophobic probe, 2-(p-toluidino)naphthalene-6-sulfonate, was observed upon incubating with the CaMLD and further increased by Ca²⁺. The Ca²⁺-dependent enhancement of fluorescence intensity was strongly suppressed by the wild type oligopeptide, but not by the mutant one. Kinetic experiments were performed with BIAcore™ where binding of the CaMLD to the oligopeptide immobilized on a biosensor chip was detected as real time signals of surface plasmon resonance. The determined dissociation constant (K_D) was 3.1 × 10^-9 M. These results suggest that the region A of calpastatin binds to the CaMLD in a specific manner similar to interactions between calmodulin-binding peptides and calmodulin where hydrophobic properties are known to be important.