Roles of individual EF-hands in the activation of m-calpain by calcium

Previn Dutt, J. Simon C. Arthur, Pawel Grochulski, Miroslaw Cygler, John S. Elce (Lead / Corresponding author)

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    38 Citations (Scopus)


    m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca2+-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca2+-binding EF-hands in each subunit, but their relative affinities for Ca2+ are not known. In the present study mutations were made in both subunits to reduce the Ca2+-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 Ca2+ 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 Ca2+ were otherwise identical to that of the Ca2+-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca2+ requirement (K(d)) was approx. 350 μM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in Kd, 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 Kd 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 Ca2+ and provides the major driving force for conformational change.

    Original languageEnglish
    Pages (from-to)37-43
    Number of pages7
    JournalBiochemical Journal
    Issue number1
    Publication statusPublished - 9 May 2000


    • Calcium binding
    • Co-operativity
    • Crystallography
    • Hill plot
    • Site-directed mutagenesis

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology


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