Structural Determinants of the Calpain Inhibitory Activity of Calpastatin Peptide B27-WT

• Published in: The Journal of biological chemistry 2003-03, Vol.278 (10), p.7800-7809
• Main Authors: Betts, Russell, Weinsheimer, Shantel, Blouse, Grant E, Anagli, John
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calcium-Binding Proteins - chemistry; Calpain - antagonists & inhibitors; Cysteine Proteinase Inhibitors - chemistry; Cysteine Proteinase Inhibitors - pharmacology; Humans; Kinetics; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Protein Conformation; Sequence Homology, Amino Acid; Swine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M208350200

Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of μ- and m -calpain. In this report, a combination of β-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two “hot spots,” Leu 11 -Gly 12 and Thr 17 -Ile 18 -Pro 19 , in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu 11 -Gly 12 and Thr 17 -Ile 18 -Pro 19 backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.