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A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus

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Abstract

Bisphosphorylation of two adjacently located serine residues in the heart-specific N-terminus of the cTnI subunit reduces calcium affinity of the cTnC subunit. An interaction of the phosphorylation region of cTnI with acidic residues of another cTn subunit has been proposed formerly based on 31P nuclear magnetic resonance (NMR) data. A possible candidate is cTnC. Thus, an interaction model of cTnC with the bisphosphorylated cTnI N-terminus has been built using a homology model of hcTnC based on the crystal structure of tusTnC and the structure of the phosphorylation region of cTnI determined by 2D NMR. By computational search, five clusters of acidic residues on cTnC might interact with the cTnI phosphorylation region. Three sites could be excluded by 31P-NMR experiments. The two remaining sites are located in the N-terminal helix of cTnC and between calcium binding sites III and IV. Reorientation of the arginine and phosphoserine sidechains within the␣phosphorylation region as proposed by refined docking could explain the formerly measured changes in pKa app values. Thus, local pKa changes might lead to the reduction of calcium affinity observed upon cTnI bisphosphorylation.

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References

  • AL-HILLAWI, E., BHANDARI, D. G., TRAYER, R. H. & TRAYER, I. P. (1995) The effects of phosphorylation of cardiac troponin I on its interaction with actin and cardiac troponin. Eur. J. Biochem. 228, 962–70.

    PubMed  Google Scholar 

  • ARDELT, P., DORKA, D., JAQUET, K., HEILMEYER, L. M. G., JR., KÖRTKE, H., KÖRFER, R. & NOTOHAMIPRODJO, G. (1998) Microanalysis and distribution of cardiac troponin I phospho species in heart areas. Biol. Chem. 379, 341–7.

    PubMed  Google Scholar 

  • BABU, A., SU, H., RYU, Y. & GULATI, J. (1992) Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination by genetic engineering. J. Biol. Chem. 267, 15469–74.

    PubMed  Google Scholar 

  • BAX, A. & DAVIS, D. G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355–60.

    Google Scholar 

  • BEIER, N., JAQUET, K., SCHNACKERZ, K. & HEILMEYER, L. M. G., JR. (1988) Isolation and characterization of a highly phosphorylated troponin from bovine heart. Eur. J. Biochem. 176, 327–34.

    PubMed  Google Scholar 

  • BIDLINGMEYER, B. A., COHEN, S. A. & TARVIN, T. L. (1984) Rapid analysis of amino acids using pre-column derivatization. J. Chromat. 336, 93–104.

    Google Scholar 

  • COLLINS, J. H. (1991) Myosin light chains and troponin C: structural and evolutionary relationships revealed by amino acid comparisons. J. Muscle Res. Cell Motil. 12, 3–25.

    PubMed  Google Scholar 

  • DAVIS, D. G. & BAX, A. (1985) Assignment of complex 1H spectra via two-dimensional homonuclear Hartmann-Hahn spectroscopy. J. Am. Chem. Soc. 107, 2820–21.

    Google Scholar 

  • DAYHOFF, M. O., BARKER, W. C. & HUNT, L. T. (1983) Establishing homologies in protein sequences. Methods Enzymol. 91, 524–45.

    PubMed  Google Scholar 

  • ENGLAND, P. (1976) Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart. Biochem. J. 160, 295–304.

    PubMed  Google Scholar 

  • ERNST, R. R., BODENHAUSEN, G. & WOKAUN, A. (1987) Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Oxford: Clarendon Press.

    Google Scholar 

  • FARAH, C. S. & REINACH, F. C. (1995) The troponin complex and regulation of muscle contraction. FASEB J. 9, 755–67.

    PubMed  Google Scholar 

  • FORSEN, S., KORDEL, J., GRUNDSTROM, T. & CHAZIN, W. J. (1993) The molecular anatomy of a calcium binding protein. Acc. Chem. Res. 26, 7–14.

    Google Scholar 

  • GAHLMANN, R., WADE, R. & GUNNING, P. (1988) Differential expression of slow and fast skeletal muscle troponin C is expressed in human fibroblasts. J. Mol. Biol. 201, 379–91.

    PubMed  Google Scholar 

  • GULATI, J., BABU, A. & SU, H. (1992) Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle with genetically engineered cardiac-skeletal chimeric troponin C. J. Biol. Chem. 267, 25073–7.

    PubMed  Google Scholar 

  • GREASER, M. L. & GERGELY, J. (1971) Reconstitution of troponin activity from three protein components. J. Biol. Chem. 246, 4226–33.

    PubMed  Google Scholar 

  • HERBERG, F. W., BELL, S. M. & TAYLOR, S. S. (1993) Expression of the catalytic subunit of cAMP dependent protein kinase in Escherichia coli: multiple isoenzymes reflect different phosphorylation states. Protein Eng. 6, 771–7.

    PubMed  Google Scholar 

  • HERZBERG, O. & JAMES, M. N. G. (1988) Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 Å resolution. J. Mol. Biol. 203, 761–79.

    PubMed  Google Scholar 

  • JAQUET, K., KORTE, K., SCHNACKERZ, K., VYSKA, K. & HEILMEYER, L. M. G., JR. (1993) Characterization of the cardiac troponin I phosphorylation domain by 31P-NMR spectroscopy. Biochemistry 32, 13873–8.

    PubMed  Google Scholar 

  • JAQUET, K., THIELECZEK, R. & HEILMEYER, L. M. G., JR. (1995) Pattern formation on cardiac troponin I by consecutive phosphorylation and dephosphorylation. Eur. J. Biochem. 231, 468–90.

    Google Scholar 

  • LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–85.

    PubMed  Google Scholar 

  • MARION, D. & WÜTHRICH, K. (1983) Application of phase sensitive two dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun. 113, 967–74.

    PubMed  Google Scholar 

  • MCKILLOP, D. F. A. & GEEVES M. A. (1991) Regulation of the actomyosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two actomyosin subfragment 1 states. Biochem. J. 279, 711–18.

    PubMed  Google Scholar 

  • MEYER, H. E., HOFFMANN-POSORSKE, E. & HEILMEYER, L. M. G., JR. (1991) Determination and location of phosphoserine in proteins and peptides by conversion to S-ethylcysteine. Meth. Enzymol. 201, 169–85.

    PubMed  Google Scholar 

  • MITTMANN, K., JAQUET, K. & HEILMEYER, L. M. G., JR (1990) A common motif of two adjacent phosphoserines in bovine, rabbit and human cardiac troponin. FEBS Lett. 273, 41–5.

    PubMed  Google Scholar 

  • MITTMANN, K., JAQUET, K. & HEILMEYER, L. M. G., JR (1992) Ordered phosphorylation of a duplicated minimal recognition motif for cAMP-dependent protein kinase present in cardiac troponin I. FEBS Lett. 302, 133–7.

    PubMed  Google Scholar 

  • OLAH, G. A. & TREWHELLA, J. (1994) A model structure of the muscle protein complex 4Ca2+ troponin C. troponin I derived from small-angle scattering data: implications for regulation. Biochemistry 33, 12800–806.

    PubMed  Google Scholar 

  • OVASKA, M. & TASKINEN, J. (1991) A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs. Proteins: Structure, Function, and Genetics 11, 79–94.

    Google Scholar 

  • PLATEAU, P. & GUERON, M. (1982) Exchangeable proton-NMR without baseline distortion using new strong-pulse sequences. J. Am. Chem. Soc. 104, 7310–11.

    Google Scholar 

  • POTTER, J. D. (1982) Preparation of troponin and its subunits. Methods Enzymol. 84, 241–63.

    Google Scholar 

  • QUIRK, P. G., PATCHELL, V. B., GAO, Y., LEVINE, B. A. & PERRY, S. V. (1995) Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin I. Structure-activity implications of ordered phosphorylation. FEBS Lett. 370, 175–8.

    PubMed  Google Scholar 

  • RAREY, M., KRAMER, B., LANGAUER, T. & KLEBE, G. (1996) A fast flexible docking method using an incremental construction algorithm. J. Mol. Biol. 26, 470–89.

    Google Scholar 

  • REIFFERT, S. U., JAQUET, K., HEILMEYER, L. M. G., JR., RITCHIE, M. D. & GEEVES, M. A. (1996) Bisphosphorylation of cardiac troponin I modulates the Ca(2+)-dependent binding of myosin subfragment 1 to reconstituted thin filaments. FEBS Lett. 384, 43–7.

  • REIFFERT, S. U., JAQUET, K., HEILMEYER, L. M. G., JR & HERBERG, F. W. (1998) Stepwise subunit interaction changes by mono and bisphosphorylation of cardiac troponin I. Biochemistry, in press.

  • ROSENFELD, R., VAJDA, S. & DELISA, C. (1995) Flexible docking and design. Ann. Rev. Biophys. Biomol. Struc. 24, 677–700.

    Google Scholar 

  • ROSKOSKI, R., JR. (1983) Assay of protein kinase. Meth. Enzymol. 99, 3–7.

    PubMed  Google Scholar 

  • SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edn) Cold Spring Harbor, NY: Laboratory Press.

    Google Scholar 

  • SIA, S. K., LI, M. X., SPYRACOPOULOS, L., GAGNE, S. M., LIU, W., PUTKEY, J. A. & SYKES, B. D. (1997) Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain J. Biol. Chem. 272, 18216–21.

    PubMed  Google Scholar 

  • SMITH, L., GREENFIELD, N. J. & HITCHCOCKDEGREGORI, S. E. (1994) The effects of deletion of the amino-terminal helix on troponin C function and stability. J. Biol. Chem. 269, 9857–63.

    PubMed  Google Scholar 

  • STULL, J. T. & BUSS, J. E. (1977) Phosphorylation of cardiac troponin I by cyclic adenosine 3':5' monophosphate dependent protein kinase. J. Biol. Chem. 252, 851–7.

    PubMed  Google Scholar 

  • SWEENEY, H. L., BRITO, R. M. M., ROSEVEAR, P. R. & PUTKEY, J. A. (1990) Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Proc. Natl Acad. Sci. USA 87, 9538–42.

    PubMed  Google Scholar 

  • SWIDEREK, K., JAQUET, K., MEYER, H. E. & HEILMEYER, L. M. G., JR. (1988) Cardiac troponin I, isolated from bovine heart, contains two adjacent phosphoserines. Eur. J. Biochem. 176, 335–42.

    PubMed  Google Scholar 

  • VALLINS, W. J., BRAND, N. J., DABHADE, N., BUTLERBROWNE, G., YACOUG, M. H. & BARTON, P. J (1990) Molecular cloning of human cardiac troponin I using polymerase chain reaction. FEBS Lett. 270, 57–61.

    PubMed  Google Scholar 

  • VILLAR-PALASI, C. & KUMON, A. (1981) Purification and properties of dog cardiac troponin T kinase. J. Biol. Chem. 256, 7409–15.

    PubMed  Google Scholar 

  • WÜTHRICH, K. (1986) Polypeptide secondary structures in proteins by NMR. In NMR of Proteins and Nucleic Acids, pp. 162–75. Chichester: John Wiley.

    Google Scholar 

  • XU, G. Q. & HITCHCOCK-DEGREGORI, S. E. H. (1988) Synthesis of a troponin C cDNA and expression of wild-type and mutant proteins in Escherichia coli. J. Biol. Chem. 263, 13962–7.

    PubMed  Google Scholar 

  • ZHANG, R., ZHAO, J. & POTTER, J. D. (1995a) Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C. J. Biol. Chem. 270, 30773–80.

    PubMed  Google Scholar 

  • ZHANG, R., ZHAO, J., MANDVENO, A. & POTTER, J. D. (1995b) Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ. Res. 76, 1028–35.

    PubMed  Google Scholar 

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Authors
  1. Kornelia Jaquet
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  2. Karin Lohmann
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  3. Thaddäus Holak
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  4. Michael Czisch
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  5. Jagdish Gulati
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  6. Ralph Jaquet
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Jaquet, K., Lohmann, K., Holak, T. et al. A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus. J Muscle Res Cell Motil 19, 647–659 (1998). https://doi.org/10.1023/A:1005381131102

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