Roles of individual EF-hands in the activation of m-calpain by calcium.
m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.
Dutt
P
P
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Arthur
J S
JS
Grochulski
P
P
Cygler
M
M
Elce
J S
JS
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D020832:EF Hand Motifs / Q000502:physiology*; D004789:Enzyme Activation; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D013725:Terbium / Q000494:pharmacology; D017186:Titrimetry
D013725:Terbium; D002154:Calpain; D002118:Calcium