Nature Structural Biology
4, 539 - 547 (1997)
doi:10.1038/nsb0797-539
Crystal structure of calcium bound domain VI of calpain at 1.9 Å resolution and its role in enzyme assembly, regulation, and inhibitor bindingGuang-da Lin1, Debasish Chattopadhyay1, Masatoshi Maki2, Kevin K. W. Wang3, Mike Carson1, Lei Jin1, Po-wai Yuen5, Emiko Takano4, Masakazu Hatanaka4, Lawrence J. DeLucas1, 6
& Sthanam V.L. Narayana1, 6, 7
1Center for Macromolecular Crystallography, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
2School of Agriculture, Nagoya University, Nagoya, Japan.
3Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Warner Lambert Company, USA.
4Institute for Virus Research, Kyoto University, Kyoto, Japan.
5Medicinal Chemistry, Parke-Davis Pharmaceutical Research, Warner Lambert Company, USA.
6School of Optometry, University of Alabama at Birmingham, USA.
7email: narayana@orion.cmc.uab.edu 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.
REFERENCES
- 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). | Article | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ChemPort |
- Zhang, W.& Mellgren, R.L. Calpain subunits remain associated during catalysis. Biochem. Biophys. Acta. 227, 890−896 (1996). | Article | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- Reddy, M.K., Etlinger, J.D., Rabinowitz, M., Fischman, D.A., Zak, R. Removal of Z-lines and a
 -actinin from isolated myobfibrils by a calcium-activated neutral proteinase. J. Biol. Chem. 250, 4278−4284 (1975). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- Wang, W.K.K., & Yuen, P.W Calpain inhibition: an overview of its therapeutic potential. Trends Pharmacol. Sci. 15, 412−419 (1994) | Article | PubMed | ISI |
- 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). | PubMed | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ChemPort |
- 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).
- 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). | Article | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- Kretsinger, R.H. EF-hands reach out. Nature Struct. Biol. 3, 12−15 (1996). | PubMed | ISI | ChemPort |
- Szebenyl, D.M., Obendorf, S.K.& Moffat, K. Structure of vitamin D-dependent calcium-binding protein from bovine intestine. Nature 294, 327−333 (1981). | PubMed |
- da Silva, A. C. R. & Reinach, F. C Calcium binding induces conformational changes in muscle regulatory proteins. Trends Biochem. Sci., 16, 53−57 (1991). | Article | PubMed |
- Ikura, M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem. Sci. 21, 14−17 (1996). | Article | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- Suzuki, K., Tsuji, S. & Ishiura, SEffect of Ca2+ on the inhibition of calcium-activated neutral protease bu leupeptin, antipain and epoxysuccinate derivatives. FEBS Lett. 136, 119−122 (1981). | Article | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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
 -calpain large subunit. J. Biol. Chem., 269, 18977−18984 (1994). | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- Chakrabarti, P. An assessment of the effect of the helix dipole in protein structures. Prot. Engng 7, 471−474 (1994). | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- Kapprell, Hans-peter., & Goll, D.E. Effects of Ca2+ on binding of the calpains and calpastatin. J. Biol. Chem. 264, 30, 17888−17896 (1989). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- Suzuki, K.& Ohno, S. Calcium activated neutral protease structure-function relationaship and functional implications. (1990) Cell Struct. Funct. 15, 1−6 (1990). | PubMed | ISI | ChemPort |
- 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). | ChemPort |
- 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). | PubMed | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | ChemPort |
- 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). | PubMed | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- Pontremoli, S. & Melloni, E. Extralysosomal protein degradation. Annu. Rev. Biochem. 55, 455−481 (1986). | Article | PubMed | ISI | ChemPort |
- 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). | PubMed |
- 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). | PubMed | ISI | ChemPort |
- 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). | PubMed | ISI | ChemPort |
- 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). | Article | PubMed | ISI | ChemPort |
- 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). | Article | ISI | ChemPort |
- Duncan, E.M. Prectical Protein Crystallography. (Academic Press Inc, London; 1993).
- Sack, J.S. CHAIN- A Crystallographic Modeling Program. J. Mol. Graphics., 6, 224 (1988). | Article | ISI |
- 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). | ChemPort |
- Brunger, A.T. X-PLOR manual Version 3.1 Yale Univ. New Haven. CT. USA (1992).
- Otwinowski, Z.. in Data Collection & Processing (eds Sawyer, L., Isaac, N. W. & Bailey, 5) 55−62. Daresbury Laboratory, Warrington (1993).
- Collaborative Computational Project, Number 4 The CCP4 suite. Programs for protein crystallography. Acta Crystallogr D50, 760−763 (1994).
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