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Crystal structure of calcium bound domain VI of calpain at 1.9 Å resolution and its role in enzyme assembly, regulation, and inhibitor binding

Abstract

The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 Å resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an ‘open’ conformation and the other (EF3-EF4) a ‘closed’ conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a ‘closed’ conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EFS's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI–inhibitor (PD150606) complex has been refined to 2.1 Å resolution. A possible mode for calpain inhibition is discussed.

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References

  1. Reddy, A. S. N., Safadi, F., Beyette, J. R., & Mykles, D. L. Calcium dependent proteinase activity in root cultures of Arabidopsis. Biochem. Biophys. Res. Commn. 199, 1089–1095 (1994).

    CAS  Google Scholar 

  2. Mellor, G. W., Sreedharan, S. k., Kowlessur, D., Thomas, E. W. & Brocklehurst, K. Catalytic-site characteristics of the porcine calpain II 80 kDa/18kDa hetrodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for catalytic site Cys/His interactive system and an ionizing modulatory group. Biochem. J. 290, 75–83 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Thompson, V.F., Goll, E.D. & Kleese, W.C. Effects of autolysis on the catalytic properties of the calpains. Biol. Chem. Hoppe-seyler, 371, Suppl. 177–185 (1990).

    CAS  PubMed  Google Scholar 

  4. Yoshizawa, T., Sorimach, H., Tomioka, S., Ishiura, S. & Suzuki, K. Calpain dissociates into subunits in the presence of calcium ions. Biochem. Biophys. Res. Commun. 208, 376–383 (1995).

    CAS  PubMed  Google Scholar 

  5. Saido, T.C., Nagao, S., Shiramine, M., Tsukaguchi, M., Yoshizawa, T., Sorimachi, H., Ito, H., Tsuchiya, T., Kawashima, S., Suzuki, K. Distinct kinetics of subunit autolysis in mammalian m-calpain activation. FEBS Lett. 346; 263–267 (1994).

    CAS  PubMed  Google Scholar 

  6. Mellgren, R.L. & Lane, R.D. Myocardial calpain 2 is inhibited by monoclonal antibodies specific for the small noncatalytic subunit. Biochem. Biophys. Acta. 954, 154–160 (1988).

    CAS  PubMed  Google Scholar 

  7. Zhang, W. & Mellgren, R.L. Calpain subunits remain associated during catalysis. Biochem. Biophys. Acta. 227, 890–896 (1996).

    CAS  Google Scholar 

  8. Busch, W.A., Stromer, M.H., Goll, D.E. & Suzuki, A. Ca2+ specific removal of Z lines from rabbit skeletal muscle. J. Cell. Biol. 52, 367–381 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Dayton, W.R., Goll, D.E., Zeece, M.G., Robson, R.M. & Reville, W.J. A Ca2+ activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle. Biochemistry, 15, 2150–2158 (1976).

    CAS  PubMed  Google Scholar 

  10. Kamakura, K., Ishura, S., Sugita, H., & Toyokura, Y. Identification of Ca2+ activated neutral protease in the peripheral nerve and its effects on neurofilament degradation. J. Neurochem. 40, 908–913 (1983).

    CAS  PubMed  Google Scholar 

  11. Nixon, R.A., Brown, B.A. & Marotta, C.A. Limited proteolytic modification of a neurofilament protein involves a proteinase activated by endogenous levels of calcium. Brain. Res., 275, 384–388 (1983).

    CAS  PubMed  Google Scholar 

  12. Reddy, M.K., Etlinger, J.D., Rabinowitz, M., Fischman, D.A., Zak, R. Removal of Z-lines and a α-actinin from isolated myobfibrils by a calcium-activated neutral proteinase. J. Biol. Chem. 250, 4278–4284 (1975).

    CAS  PubMed  Google Scholar 

  13. Tallant, E.A., Brumley, L.M., Wallace, R.W. Activation of a calmodulin-dependent phosphyatase by a Ca2+ dependent protease. Biochemistry, 27, 2205–2211 (1988).

    CAS  PubMed  Google Scholar 

  14. Wang, W.K.K., & Yuen, P.W Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. 15, 412–419 (1994)

    CAS  PubMed  Google Scholar 

  15. Lee, K.S., Frank, S., Venderklish, P., Arai, A., & Lynch, G. Inhibition of proteolysis protects hippocampal neurons from ischemia., Proc. Natl. Acad. Sci. USA 88, 7233–7237 (1991).

    CAS  PubMed  Google Scholar 

  16. Bartus, R.T., Hayward, N.J., Elliot, P.J., Sawyer, S.D., Baker, K.L., Dean, R.L., Akiyama, A., Straub, J. A., Harbeson, S. L., Li. Z., & Powers, J., Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration., Stroke 25, 2265–2270 (1994).

    CAS  PubMed  Google Scholar 

  17. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Bourg, N., Brenguir, L., Devaud, C., Pasturaud, P., Roudaut, C., Hillaire, D., Passos-Bueno, M., Zatz, M., Tischfield, J.A., Fardeau, M., Jackson, C.E., Cohen, D. & Beckmann, J.S. Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell, 81, 27–40 (1995).

    CAS  PubMed  Google Scholar 

  18. Wang, K.K.W., Nath, R., Posner, A., Raser, K.J., Buroker-Kilgore, M., Ye, Q., Takano.,E., Hatanaka, M., Maki., M., Marcoux, F.W., Caner, H., Collins, J. L., Fergus, A., Lee, K. S., Lunney, E. A., Hays, S. J., & Yuen, P.W. Alpha-Mercaptoacrylic Acid Derivatives are Selective Non-Peptide and calcium-Binding Domain-targeting calpain Inhibitors. Proc. Natl. Acad. Sci. USA 93, 6687–6692 (1996).

    CAS  PubMed  Google Scholar 

  19. Lin, G., Chattopadhyay, D., Maki, M., Takano, E., Hatanaka, M., DeLucas, L.J., Narayana, S.V.L. Purifaction, Crystallization and Preliminary X-ray diffraction studies of recombinant calcium binding domain of porcine calpain small subunit. Acta. Crystallogr. in the press (1997).

  20. Emori, Y.S., Ohno, S., Tobita, M. & Suzuki, K. Gene structure of calcium dependent protease retains the ancestral organization of the calcium-binding protein gene. FEBS Lett., 194, 249–252 (1986).

    CAS  PubMed  Google Scholar 

  21. Minami, Y., Emori, Y., Imajoh-Ohmi, S., Kawasaki, H, & Suzuki, K. Carboxy-terminal truncation and site-directed mutagenesis of the EF hand structure domain of the small subunit of rabbit calcium-dependent protease. J. Biochem., 104, 927–933 (1988).

    CAS  PubMed  Google Scholar 

  22. Ohno, S.Y., Emori, Y., Imajoh, S., Kaeasaki, H., Kisaragi, M. & Suzuki, K. Evolutionary origin of calcium-dependent protease by fusion of genes for a thiol protease & a calcium binding protein? Nature 312, 566–570 (1984).

    CAS  PubMed  Google Scholar 

  23. Kretsinger, R.H. EF-hands reach out. Nature Struct. Biol. 3, 12–15 (1996).

    CAS  PubMed  Google Scholar 

  24. Szebenyl, D.M., Obendorf, S.K. & Moffat, K. Structure of vitamin D-dependent calcium-binding protein from bovine intestine. Nature 294, 327–333 (1981).

    Google Scholar 

  25. da Silva, A. C. R. & Reinach, F. C Calcium binding induces conformational changes in muscle regulatory proteins. Trends Biochem. Sci., 16, 53–57 (1991).

    PubMed  Google Scholar 

  26. Ikura, M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem. Sci. 21, 14–17 (1996).

    CAS  PubMed  Google Scholar 

  27. Zhang, M., Tanaka, T. & Ikura, M. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature Struct. Biol. 2, 758–767 (1995).

    CAS  Google Scholar 

  28. Kuboniwa, H., Tjandra, N., Grzesiek, S., Ren, H., Klee, C.B., Bax, Ad. Solution structure of calcium-free calmodulin. Nature Struct. Biol, 2, 768–776 (1995).

    CAS  PubMed  Google Scholar 

  29. Finn, B., Evenas, J., Drakenberg, T., Waltho, J P., Thulin, E., Forsen, S. Calcium-induced structural changes and domain autonomy in calmodulin. Nature Struct. Biol, 2, 777–783 (1995).

    CAS  Google Scholar 

  30. Suzuki, K., Tsuji, S . & Ishiura, S Effect of Ca2+ on the inhibition of calcium-activated neutral protease bu leupeptin, antipain and epoxysuccinate derivatives. FEBS Lett. 136, 119–122 (1981).

    CAS  PubMed  Google Scholar 

  31. Ma, H., Yang, H.Q., Takano, E., Lee, W.J., Hatanaka, M., & Maki, M. Requirement of different subdomains of calpastatin for calpain inhibition and for binding to the calmodulin-like domains. J. Biochem. 113, 591–599 (1993).

    CAS  PubMed  Google Scholar 

  32. Yang, H.Q., Ma, H., Takanao, E., Hatanaka, M., & Maki, M. Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of μ-calpain large subunit. J. Biol. Chem., 269, 18977–18984 (1994).

    CAS  PubMed  Google Scholar 

  33. Takanao, E., Ma, H., Yang, H.Q., Maki, M., Hatanaka, M. Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains. FEBS Lett. 362, 93–97 (1995).

    Google Scholar 

  34. Chakrabarti, P. An assessment of the effect of the helix dipole in protein structures. Prot. Engng 7, 471–474 (1994).

    CAS  Google Scholar 

  35. Strynadka, N.C.J., & James, M.N.G. Crystal structures of the hekix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58, 951–998 (1989).

    CAS  PubMed  Google Scholar 

  36. Kapprell, Hans-peter., & Goll, D.E. Effects of Ca2+ on binding of the calpains and calpastatin. J. Biol. Chem. 264, 30, 17888–17896 (1989).

    CAS  PubMed  Google Scholar 

  37. Kawasaki, H., Imajoh, S., Kawashima, S., Hayashi, T. & Suzuki, K. The small subunits of calcium dependent proteases with different calcium sensitivity are identical. J. Biochem. 99, 1525–1532 (1986).

    CAS  PubMed  Google Scholar 

  38. Suzuki, K. & Ohno, S. Calcium activated neutral protease structure-function relationaship and functional implications. (1990) Cell Struct. Funct. 15, 1–6 (1990).

    CAS  PubMed  Google Scholar 

  39. Carson, M., Bugg, C.E., DeLucas, L.J. & Narayana, S.V.L. Comaprision of Homology methods with the experimental structure of a novel serine protease. Acta. Crystallogr. D50, 889–899 (1994).

    CAS  Google Scholar 

  40. Imajoh, S., Kawasaki, H. & Suzuki, K. The COOH-terminal EF-hand of calcium-activated neutral protease (CANP) is important for the association of subunits and resulting proteolytic activity. J. Biochem (Tokyo), 101, 447–452 (1987).

    CAS  Google Scholar 

  41. Coolican, S.A., Haiech, J. & Hathaway, D.R. The role of subunit autolysis in activation of smooth muscle Ca2+ dependent protease. J. Biol. Chem. 261, 4170–4176 (1986).

    CAS  PubMed  Google Scholar 

  42. Shearer, T.R., Azuma, M., David, L.L. & Murachi, T. Amelioration of cataract and proteolysis in cultured lenses by calpain inhibitor E64. Invest. Opthol. Vis. Sci. 32, 533–540 (1991).

    CAS  Google Scholar 

  43. Lee, K.S., Frank, S., Venderklish, P., Arai, A. & Lynch, G. Inhibition of proteolysis protects hippocampal neurons from ischemia., Proc. Natl. Acad. Sci. USA 88, 7233–7237 (1991).

    CAS  PubMed  Google Scholar 

  44. Bartus, R.T., Hayward, N.J., Elliot, P.J., Sawyer, S.D., Baker, K.L., Dean, R.L., Akiyama, A., Straub, J.A., Harbeson, S.L., Li, Z., & Powers, J., Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration. Stroke 25, 2265–2270 (1994).

    CAS  PubMed  Google Scholar 

  45. Pontremoli, S. & Melloni, E. Extralysosomal protein degradation. Annu. Rev. Biochem. 55, 455–481 (1986).

    CAS  PubMed  Google Scholar 

  46. Shoji, K.Y., Senshu, M., Iwashita, S., & Imahori, K. Thiol protease-specific inhibitor E-64 arrests human epidermal carcinoma A431 cells at mitotic metaphase. Proc. Natl. Acad. Sci. USA 85, 146–150 (1988).

    Google Scholar 

  47. Cook, W.J., Walter, L.J., & Walter, M.R. Drug binding of calmodulin: Crystal structure of a calmodulin-Trifluoperazine complex. Biochemistry. 33, 15259–15265 (1994).

    CAS  PubMed  Google Scholar 

  48. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G., Klee, C.B., & Bax, A. (1992) Solution structure of calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992).

    CAS  Google Scholar 

  49. Chin, D. & Means, R.A. Methionine to glutamine substitutions in the C-terminal domian of calmodilin impair the activation of threee protein kinases. J. Biol. Chem. 271, 30465–30471 (1996).

    CAS  PubMed  Google Scholar 

  50. Howard, A.J., Gilliland, G.L., Finzel, B.C., Poulos, T.L., Ohelendorf, D.H. & Salemme, F.R. J. Appl. Crystallogr. 20, 383–388 (1987).

    CAS  Google Scholar 

  51. Duncan, E.M. Prectical Protein Crystallography. (Academic Press Inc, London; 1993).

    Google Scholar 

  52. Sack, J.S. CHAIN- A Crystallographic Modeling Program. J. Mol. Graphics., 6, 224 (1988).

    Google Scholar 

  53. Jones, T.A., Zou, J.Y. & Cowman, S.W. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta. Crystallogr. A47, 110–119 (1991).

    CAS  Google Scholar 

  54. Brunger, A.T. X-PLOR manual Version 3.1 Yale Univ. New Haven. CT. USA (1992).

    Google Scholar 

  55. Otwinowski, Z. . in Data Collection & Processing (eds Sawyer, L., Isaac, N. W. & Bailey, 5) 55–62. Daresbury Laboratory, Warrington (1993).

    Google Scholar 

  56. Collaborative Computational Project, Number 4 The CCP4 suite. Programs for protein crystallography. Acta Crystallogr D50, 760–763 (1994).

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Correspondence to Sthanam V.L. Narayana.

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Lin, Gd., Chattopadhyay, D., Maki, M. et al. Crystal structure of calcium bound domain VI of calpain at 1.9 Å resolution and its role in enzyme assembly, regulation, and inhibitor binding. Nat Struct Mol Biol 4, 539–547 (1997). https://doi.org/10.1038/nsb0797-539

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