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 inter action with its endogenous inhibitor, calpastatin. The mechanism of in hibition of calpain by calpastatin has remained unsolved, although Nis himura and Goll [J. Biol. Chem. 266, 11842-11850 (1991)] reported that autolyzed calpain fragments containing calmodulin-like domains (CaMLD s) bound to an immobilized calpastatin column. We investigated the cor relation between CaMLD-binding and calpain inhibition using immobilize d columns of gene-engineered CaMLDs derived from the human mu-calpain large subunit and various recombinant calpastatin mutants. Among the f our internally repetitive inhibitory domains of calpastatin, each havi ng conserved regions A, B, and C, only domains 1 and 4 showed the bind ing 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 oligop eptide of region B with moderate calpain inhibition activity did not b ind to the column. Domain 3 acquired the binding ability on substituti on of region A with that of domain 1. These results suggest that calpa in inhibition and binding to the CaMLDs are not correlated or mediated by different subdomains of calpastatin.