title: Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis.
abstract: Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.
authors.affiliation: Department of Biochemistry and the Protein Engineering Network of Centres of Excellence, Queen's University, Kingston, Ont. K7L 3N6, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Moldoveanu
authors.forename: C M
authors.initials: CM
authors.lastname: Hosfield
authors.forename: Z
authors.initials: Z
authors.lastname: Jia
authors.forename: J S
authors.initials: JS
authors.lastname: Elce
authors.forename: P L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011993:Recombinant Fusion Proteins; D002918:Chymotrypsin; D014357:Trypsin; D002154:Calpain; D008274:Magnesium; D002118:Calcium