Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interacting with calmodulin-like domain of the proteinase.
Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca(2+)-dependent proteinase) and is known to interact with the calmodulin-like domain (CaMLD) of the proteinase in a Ca(2+)-dependent fashion. The calpastatin molecule consists of four inhibitory domains (domains 1-4) with mutually homologous sequences in three regions designated as A, B, and C. Acidic amphiphilic alpha-helical motifs are found in both regions A and C. We investigated the correlation between the calpain inhibition potency and the ability of calpastatin to bind to recombinant CaMLD of the mu-calpain large subunit using various mutant proteins of pig calpastatin domain 1 expressed in Escherichia coli. Substitution of conserved Leu-161 with Pro in region A caused a reduction in activity of both calpain inhibition and CaMLD binding. Additional substitution of Leu-236 with Pro in region C further decreased the calpain inhibitory activity and caused a loss of CaMLD binding ability. The effects of mutation in region C alone on the above activities were smaller than those in region A. Although a mutant of deletion in the entire region B had no calpain inhibitory activity, it retained the CaMLD binding ability. On the other hand, although a region B oligopeptide had a moderate inhibitory activity, it had no CaMLD binding ability. These results suggest that region A has a role in potentiating the inhibitory activity of calpastatin by interacting with CaMLD of calpain to form a tighter complex where region B exerts the inhibitory function.
Ma
H
H
Institute for Virus Research, Kyoto University, Japan.
Yang
H Q
HQ
Takano
E
E
Hatanaka
M
M
Maki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017124:Conserved Sequence; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013552:Swine
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain