Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Oct 1;367(Pt 1):263–269. doi: 10.1042/BJ20020485

Origins of the difference in Ca2+ requirement for activation of mu- and m-calpain.

Previn Dutt 1, Cherie N Spriggs 1, Peter L Davies 1, Zongchao Jia 1, John S Elce 1
PMCID: PMC1222847  PMID: 12014988

Abstract

The mu- and m-calpains are closely related Ca(2+)-dependent cysteine proteases having different in vitro Ca(2+) requirements ( K (d)), of approx. 25 and 325 microM respectively. The two isoforms are heterodimers of slightly different large (80 kDa) subunits and an identical small (28 kDa) subunit, so that the difference in K (d) values must reside in the large subunits. As assayed here, these K (d) values relate to the Ca(2+) required for the first phase of calpain activation and do not reflect the lower Ca(2+) then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding mu-type residues, but these mutations did not produce the expected decrease in K (d). In a series of hybrid (mu/m) large-subunit calpains, the K (d) values decreased progressively towards that of mu-calpain as the proportion of mu-type sequence increased from 0 to 90%. K (d) values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca(2+) binding. Nonetheless, 25% of the difference in K (d) values between mu- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

Full Text

The Full Text of this article is available as a PDF (236.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ababou A., Desjarlais J. R. Solvation energetics and conformational change in EF-hand proteins. Protein Sci. 2001 Feb;10(2):301–312. doi: 10.1110/ps.33601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ababou A., Shenvi R. A., Desjarlais J. R. Long-range effects on calcium binding and conformational change in the N-domain of calmodulin. Biochemistry. 2001 Oct 23;40(42):12719–12726. doi: 10.1021/bi010405b. [DOI] [PubMed] [Google Scholar]
  3. Arthur J. S., Mykles D. L. Calpain zymography with casein or fluorescein isothiocyanate casein. Methods Mol Biol. 2000;144:109–116. doi: 10.1385/1-59259-050-0:109. [DOI] [PubMed] [Google Scholar]
  4. Blanchard H., Grochulski P., Li Y., Arthur J. S., Davies P. L., Elce J. S., Cygler M. Structure of a calpain Ca(2+)-binding domain reveals a novel EF-hand and Ca(2+)-induced conformational changes. Nat Struct Biol. 1997 Jul;4(7):532–538. doi: 10.1038/nsb0797-532. [DOI] [PubMed] [Google Scholar]
  5. Carafoli E., Molinari M. Calpain: a protease in search of a function? Biochem Biophys Res Commun. 1998 Jun 18;247(2):193–203. doi: 10.1006/bbrc.1998.8378. [DOI] [PubMed] [Google Scholar]
  6. Dutt P., Arthur J. S., Grochulski P., Cygler M., Elce J. S. Roles of individual EF-hands in the activation of m-calpain by calcium. Biochem J. 2000 May 15;348(Pt 1):37–43. [PMC free article] [PubMed] [Google Scholar]
  7. Elce J. S. Expression of m-calpain in Escherichia coli. Methods Mol Biol. 2000;144:47–54. doi: 10.1385/1-59259-050-0:47. [DOI] [PubMed] [Google Scholar]
  8. Elce J. S., Hegadorn C., Arthur J. S. Autolysis, Ca2+ requirement, and heterodimer stability in m-calpain. J Biol Chem. 1997 Apr 25;272(17):11268–11275. doi: 10.1074/jbc.272.17.11268. [DOI] [PubMed] [Google Scholar]
  9. Elce J. S., Hegadorn C., Gauthier S., Vince J. W., Davies P. L. Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography. Protein Eng. 1995 Aug;8(8):843–848. doi: 10.1093/protein/8.8.843. [DOI] [PubMed] [Google Scholar]
  10. Fukui I., Toyohara H., Ito K., Hamakubo T., Murachi T. Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes. Biochemistry. 1988 May 3;27(9):3260–3267. doi: 10.1021/bi00409a021. [DOI] [PubMed] [Google Scholar]
  11. Goll D. E., Thompson V. F., Taylor R. G., Zalewska T. Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin? Bioessays. 1992 Aug;14(8):549–556. doi: 10.1002/bies.950140810. [DOI] [PubMed] [Google Scholar]
  12. Hosfield C. M., Elce J. S., Davies P. L., Jia Z. Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J. 1999 Dec 15;18(24):6880–6889. doi: 10.1093/emboj/18.24.6880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  14. Kretsinger R. H. EF-hands embrace. Nat Struct Biol. 1997 Jul;4(7):514–516. doi: 10.1038/nsb0797-514. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lin G. D., Chattopadhyay D., Maki M., Wang K. K., Carson M., Jin L., Yuen P. W., Takano E., Hatanaka M., DeLucas L. J. Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding. Nat Struct Biol. 1997 Jul;4(7):539–547. doi: 10.1038/nsb0797-539. [DOI] [PubMed] [Google Scholar]
  17. Meyer S. L., Bozyczko-Coyne D., Mallya S. K., Spais C. M., Bihovsky R., Kaywooya J. K., Lang D. M., Scott R. W., Siman R. Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system. Biochem J. 1996 Mar 1;314(Pt 2):511–519. doi: 10.1042/bj3140511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Moldoveanu Tudor, Hosfield Christopher M., Lim Daniel, Elce John S., Jia Zongchao, Davies Peter L. A Ca(2+) switch aligns the active site of calpain. Cell. 2002 Mar 8;108(5):649–660. doi: 10.1016/s0092-8674(02)00659-1. [DOI] [PubMed] [Google Scholar]
  19. Mäler L., Blankenship J., Rance M., Chazin W. J. Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k. Nat Struct Biol. 2000 Mar;7(3):245–250. doi: 10.1038/73369. [DOI] [PubMed] [Google Scholar]
  20. Nakagawa K., Masumoto H., Sorimachi H., Suzuki K. Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation. J Biochem. 2001 Nov;130(5):605–611. doi: 10.1093/oxfordjournals.jbchem.a003025. [DOI] [PubMed] [Google Scholar]
  21. Nelson Melanie R., Thulin Eva, Fagan Patricia A., Forsén Sture, Chazin Walter J. The EF-hand domain: a globally cooperative structural unit. Protein Sci. 2002 Feb;11(2):198–205. doi: 10.1110/ps.33302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ono Y., Sorimachi H., Suzuki K. Structure and physiology of calpain, an enigmatic protease. Biochem Biophys Res Commun. 1998 Apr 17;245(2):289–294. doi: 10.1006/bbrc.1998.8085. [DOI] [PubMed] [Google Scholar]
  23. Pal G. P., Elce J. S., Jia Z. Dissociation and aggregation of calpain in the presence of calcium. J Biol Chem. 2001 Sep 10;276(50):47233–47238. doi: 10.1074/jbc.M105149200. [DOI] [PubMed] [Google Scholar]
  24. Raser K. J., Posner A., Wang K. K. Casein zymography: a method to study mu-calpain, m-calpain, and their inhibitory agents. Arch Biochem Biophys. 1995 May 10;319(1):211–216. doi: 10.1006/abbi.1995.1284. [DOI] [PubMed] [Google Scholar]
  25. Sorimachi H., Amano S., Ishiura S., Suzuki K. Primary sequences of rat mu-calpain large and small subunits are, respectively, moderately and highly similar to those of human. Biochim Biophys Acta. 1996 Nov 11;1309(1-2):37–41. doi: 10.1016/s0167-4781(96)00135-2. [DOI] [PubMed] [Google Scholar]
  26. Sorimachi H., Suzuki K. The structure of calpain. J Biochem. 2001 May;129(5):653–664. doi: 10.1093/oxfordjournals.jbchem.a002903. [DOI] [PubMed] [Google Scholar]
  27. Strobl S., Fernandez-Catalan C., Braun M., Huber R., Masumoto H., Nakagawa K., Irie A., Sorimachi H., Bourenkow G., Bartunik H. The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):588–592. doi: 10.1073/pnas.97.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tompa P., Emori Y., Sorimachi H., Suzuki K., Friedrich P. Domain III of calpain is a ca2+-regulated phospholipid-binding domain. Biochem Biophys Res Commun. 2001 Feb 9;280(5):1333–1339. doi: 10.1006/bbrc.2001.4279. [DOI] [PubMed] [Google Scholar]
  29. Vilei E. M., Calderara S., Anagli J., Berardi S., Hitomi K., Maki M., Carafoli E. Functional properties of recombinant calpain I and of mutants lacking domains III and IV of the catalytic subunit. J Biol Chem. 1997 Oct 10;272(41):25802–25808. doi: 10.1074/jbc.272.41.25802. [DOI] [PubMed] [Google Scholar]
  30. Yap K. L., Ames J. B., Swindells M. B., Ikura M. Diversity of conformational states and changes within the EF-hand protein superfamily. Proteins. 1999 Nov 15;37(3):499–507. doi: 10.1002/(sici)1097-0134(19991115)37:3<499::aid-prot17>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES