Requirement of different subdomains of calpastatin for calpain inhibition and for binding to calmodulin-like domains.
Calpain requires Ca2+ for both proteolysis of its substrates and interaction with its endogenous inhibitor, calpastatin. The mechanism of inhibition of calpain by calpastatin has remained unsolved, although Nishimura and Goll [J. Biol. Chem. 266, 11842-11850 (1991)] reported that autolyzed calpain fragments containing calmodulin-like domains (CaMLDs) bound to an immobilized calpastatin column. We investigated the correlation between CaMLD-binding and calpain inhibition using immobilized columns of gene-engineered CaMLDs derived from the human mu-calpain large subunit and various recombinant calpastatin mutants. Among the four internally repetitive inhibitory domains of calpastatin, each having conserved regions A, B, and C, only domains 1 and 4 showed the binding activity. The region B deletion mutant of domain 1, retaining the CaMLD-binding ability, no longer had the calpain inhibition activity, and became susceptible to proteolysis. In contrast, a synthetic oligopeptide of region B with moderate calpain inhibition activity did not bind to the column. Domain 3 acquired the binding ability on substitution of region A with that of domain 1. These results suggest that calpain inhibition and binding to the CaMLDs are not correlated or mediated by different subdomains of calpastatin.
Ma
H
H
Institute for Virus Research, Kyoto University.
Yang
H Q
HQ
Takano
E
E
Lee
W J
WJ
Hatanaka
M
M
Maki
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; C017967:W 7; C033668:calpastatin; D005982:Glutathione Transferase; D002154:Calpain; D008274:Magnesium; D002118:Calcium