Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of mu-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 Ca(2+)-dependent interaction with calpastatin, specificity and molecular basis of the interaction have remained unclear. We investigated the interaction between the CaMLD of human mu-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 Ca(2+)-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 Ca(2+)-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 (KD) was 3.1 x 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.
Yang
H Q
HQ
Institute for Virus Research, Kyoto University, Japan.
Ma
H
H
Takano
E
E
Hatanaka
M
M
Maki
M
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000031:analogs & derivatives; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; C033668:calpastatin; D002154:Calpain; D002118:Calcium