Advertisement

Crystal Structures of Tropomyosin: Flexible Coiled-Coil

  • Yasushi Nitanai
  • Shiho Minakata
  • Kayo Maeda
  • Naoko Oda
  • Yuichiro Maéda
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 592)

13.1. Abstruct

Tropomyosin (Tm) is a 400 Å long coiled coil protein, and with troponin it regulates contraction in skeletal and cardiac muscles in a [Ca2+]-dependent manner. Tm consists of multiple domains with diverse stabilities in the coiled coil form, thus providing Tm with dynamic flexibility. This flexibility must play important roles in the actin binding and the cooperative transition between the calcium regulated states of the entire muscle thin filament. In order to understand the flexibility of Tm in its entirety, the atomic coordinates of Tm are needed. Here we report the two crystal structures of Tm segments. One is rabbit skeletal muscle α-Tm encompassing residues 176–284 with an N-terminal extension of 25 residues from the leucine zipper sequence of GCN4, which includes the region that interacts with the troponin core domain. The other is α-Tm encompassing residues 176–273 with N- and C-terminal extensions of the leucine zipper sequences. These two crystal structures imply that this molecule is a flexible coiled coil. First, Tm’s are not homogeneous and smooth coiled coils, but instead they undulate, with highly fluctuating local parameters specifying the coiled coil. Independent fluctuating showed by two crystal structures is important. Second, in the first crystal, the coiled coil is bent by 9 degrees in the region centered about Y214-E218-Y221, where the inter-helical distance has its maximum. On the other hand, no bend is observed at the same region in the second crystal even if its inter-helical distance has also its maximum. E218, an unusual negatively charged residue at the a position in the heptad repeat, seems to play the key role in destabilizing the coiled coil with alanine destabilizing clusters.

Keywords

Leucine Zipper Coiled Coil Heptad Repeat Helical Distance Rabbit Skeletal Muscle 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

13.7. References

  1. 1.
    K. Bailey, Tropomyosin: a new asymmetric protein component of muscle, Nature 157, 368–369 (1946).Google Scholar
  2. 2.
    S. Ebashi, Calcium ions and muscle contraction, Nature 240(5378), 217–218 (1972).PubMedCrossRefGoogle Scholar
  3. 3.
    S. Ebashi, and M. Endo, Calcium ion and muscle contraction, Prog. Biophys. Mol. Biol. 18, 123–183 (1968).PubMedCrossRefGoogle Scholar
  4. 4.
    S. V. Perry, Vertebrate tropomyosin: distribution, properties and function, J. Muscle Res. Cell Motil. 22(1), 5–49 (2001).PubMedCrossRefGoogle Scholar
  5. 5.
    J. P. Lees-Miller, and D. M. Helfman, The molecular basis for tropomyosin isoform diversity, Bioessays 13(9), 429–437 (1991).PubMedCrossRefGoogle Scholar
  6. 6.
    I. Ohtsuki, K. Maruyama, and S. Ebashi, Regulatory and cytoskeletal proteins of vertebrate skeletal muscle, Adv. Protein. Chem. 38, 1–67 (1986).PubMedCrossRefGoogle Scholar
  7. 7.
    A. N. Lupas, and M. Gruber, The structure of alpha-helical coiled coils, Adv. Protein. Chem. 70, 37–78 (2005).PubMedCrossRefGoogle Scholar
  8. 8.
    F. H. Crick, Is alpha-keratin a coiled coil?, Nature 170(4334), 882–883 (1952).PubMedCrossRefGoogle Scholar
  9. 9.
    T. Alber, Structure of the leucine zipper, Curr. Opin. Genet. Dev. 2(2), 205–210 (1992).PubMedCrossRefGoogle Scholar
  10. 10.
    D. N. Marti, and H. R. Bosshard, Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges, J. Mol. Biol. 330(3), 621–637 (2003).PubMedCrossRefGoogle Scholar
  11. 11.
    B. Tripet, K. Wagschal, P. Lavigne, C. T. Mant, and R. S. Hodges, Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position “d”, J. Mol. Biol. 300(2), 377–402 (2000).PubMedCrossRefGoogle Scholar
  12. 12.
    K. Wagschal, B. Tripet, P. Lavigne, C. Mant, and R. S. Hodges, The role of position a in determining the stability and oligomerization state of alpha-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins, Protein Sci. 8(11), 2312–2329 (1999).PubMedCrossRefGoogle Scholar
  13. 13.
    E. K. O’Shea, J. D. Klemm, P. S. Kim, and T. Alber, X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil, Science 254(5031), 539–544 (1991).PubMedCrossRefGoogle Scholar
  14. 14.
    S. M. Lu, and R. S. Hodges, Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability, Protein Sci. 13(3), 714–726 (2004).PubMedCrossRefGoogle Scholar
  15. 15.
    S. C. Kwok, and R. S. Hodges, Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils, J. Biol. Chem. 279(20), 21576–21588 (2004).PubMedCrossRefGoogle Scholar
  16. 16.
    A. Singh, and S. E. Hitchcock-DeGregori, Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding, Biochemistry 42(48), 14114–14121 (2003).PubMedCrossRefGoogle Scholar
  17. 17.
    K. I. Sano, K. Maeda, H. Taniguchi, and Y. Maeda, Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule, Eur. J. Biochem. 267(15), 4870–4877 (2000).PubMedCrossRefGoogle Scholar
  18. 18.
    E. F. Woods, The conformational stabilities of tropomyosins, Aust. J. Biol. Sci. 29(5–6), 405–418 (1976).PubMedGoogle Scholar
  19. 19.
    A. Sato, and K. Mihashi, Thermal modification of structure of tropomyosin. I. Changes in the intensity and polarization of the intrinsic fluorescence (tyrosine), J. Biochem. (Tokyo) 71(4), 597–605 (1972).Google Scholar
  20. 20.
    S. S. Lehrer, Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies, J. Mol. Biol. 118(2), 209–226 (1978).PubMedCrossRefGoogle Scholar
  21. 21.
    P. Graceffa, and S. S. Lehrer, The excimer fluorescence of pyrene-labeled tropomyosin. A probe of conformational dynamics, J. Biol. Chem. 255(23), 11296–11300 (1980).PubMedGoogle Scholar
  22. 22.
    S. L. Betcher-Lange, and S. S. Lehrer, Pyrene excimer fluorescence in rabbit skeletal alphaalphatropo-myosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation, J. Biol. Chem. 253(11), 3757–3760 (1978).PubMedGoogle Scholar
  23. 23.
    D. R. Betteridge, and S. S. Lehrer, Two conformational states of didansylcystine-labeled rabbit cardiac tropomyosin, J. Mol. Biol. 167(2), 481–496 (1983).PubMedCrossRefGoogle Scholar
  24. 24.
    P. Graceffa, and S. S. Lehrer, Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin., Biochemistry 23(12), 2606–2612 (1984).PubMedCrossRefGoogle Scholar
  25. 25.
    B. F. Edwards, and B. D. Sykes, Nuclear magnetic resonance evidence for the coexistence of several conformational states of rabbit cardiac and skeletal tropomyosins, Biochemistry 19(12), 2577–2583 (1980).PubMedCrossRefGoogle Scholar
  26. 26.
    S. A. Potekhin, and P. L. Privalov, Co-operative blocks in tropomyosin, J. Mol. Biol. 159(3), 519–535 (1982).PubMedCrossRefGoogle Scholar
  27. 27.
    D. L. Williams Jr., and C. A. Swenson, Tropomyosin stability: assignment of thermally induced conformational transitions to separate regions of the molecule, Biochemistry 20(13), 3856–3864 (1981).PubMedCrossRefGoogle Scholar
  28. 28.
    F. G. Whitby, and G. N. Phillips Jr., Crystal structure of tropomyosin at 7 Angstroms resolution, Proteins 38(1), 49–59 (2000).PubMedCrossRefGoogle Scholar
  29. 29.
    J. H. Brown, K. H. Kim, G. Jun, N. J. Greenfield, R. Dominguez, N. Volkmann, S. E. Hitchcock-DeGregori, and C. Cohen, Deciphering the design of the tropomyosin molecule, Proc. Natl. Acad. Sci. USA 98(15), 8496–8501 (2001).PubMedCrossRefGoogle Scholar
  30. 30.
    Li, Y., S. Mui, J. H. Brown, J. Strand, L. Reshetnikova, L. S. Tobacman, and C. Cohen, The crystal structure of the C-terminal fragment of striated-muscle alpha-tropomyosin reveals a key troponin T recognition site, Proc. Natl. Acad. Sci. USA 99(11), 7378–7383 (2002).PubMedCrossRefGoogle Scholar
  31. 31.
    N. J. Greenfield, G. V. Swapna, Y. Huang, T. Palm, S. Graboski, G. T. Montelione, and S. E. Hitchcock-DeGregori, The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices, Biochemistry 42(3), 614–619 (2003).PubMedCrossRefGoogle Scholar
  32. 32.
    N. Ookubo, Intramolecular disulfide linked alphabeta and alphaalpha in oxidized tropomyosin: separation, identification, and process of formation, J. Biochem. (Tokyo) 81(4), 923–931 (1977).Google Scholar
  33. 33.
    T. Shimizu, K. Ihara, R. Maesaki, M. Amano, K. Kaibuchi, and T. Hakoshima, Parallel coiled-coil association of the RhoA-binding domain in Rho-kinase, J. Biol. Chem. 278(46), 46046–46051 (2003).PubMedCrossRefGoogle Scholar
  34. 34.
    M. V. Vinogradova, D. B. Stone, G. G. Malanina, C. Karatzaferi, R. Cooke, R. A. Mendelson, and R. J. Fletterick, Ca(2+)-regulated structural changes in troponin, Proc. Natl. Acad. Sci. USA 102(14), 5038–5043 (2005).PubMedCrossRefGoogle Scholar
  35. 35.
    S. Takeda, A. Yamashita, K. Maeda, and Y. Maeda, Structure of the core domain of human cardiac troponin in the Ca(2+)-saturated form, Nature 424(6944), 35–41 (2003).PubMedCrossRefGoogle Scholar
  36. 36.
    R. Maytum, F. Bathe, M. Konrad, and M. A. Geeves, Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation, J. Biol. Chem. 279(18), 18203–18209 (2004).PubMedCrossRefGoogle Scholar
  37. 37.
    L. Kluwe, K. Maeda, A. Miegel, S. Fujita-Becker, Y. Maeda, G. Talbo, T. Houthaeve, and R. Kellner, Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions, J. Muscle Res. Cell Motil. 16(2), 103–110 (1995).PubMedCrossRefGoogle Scholar
  38. 38.
    M. Sugahara, and M. Miyano, Development of high-throughput automatic protein crystallization and observation system, Tanpakushitsu Kakusan Koso 47(8 Suppl), 1026–1032 (2002).PubMedGoogle Scholar
  39. 39.
    S. Adachi, T. Oguchi, H. Tanida, S.-Y. Park, H. Shimizu, H. Miyatake, N. Kamiya, Y. Shiro, Y. Inoue, T. Ueki, and T. Iizuka, The RIKEN Structural Biology Beamline II (BL44B2) at the SPring-8, Nucl. Instrum. Methods Phys. Res. A 467, 711–714 (2001).Google Scholar
  40. 40.
    J. W. Pflugrath, The finer things in X-ray diffraction data collection, Acta Crystallogr. D Biol. Crystallogr. 55(Pt 10), 1718–1725 (1999).PubMedCrossRefGoogle Scholar
  41. 41.
    J. Navaza, Implementation of molecular replacement in AMoRe, Acta Crystallogr. D Biol. Crystallogr. 57(Pt 10), 1367–1372 (2001).PubMedCrossRefGoogle Scholar
  42. 42.
    E. Potterton, P. Briggs, M. Turkenburg, and E. Dodson, A graphical user interface to the CCP4 program suite, Acta Crystallogr. D Biol. Crystallogr. 59(Pt 7), 1131–1137 (2003).PubMedCrossRefGoogle Scholar
  43. 43.
    Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography, Acta Crystallogr. D Biol. Crystallogr. 50(Pt 5), 760–763 (1994).CrossRefGoogle Scholar
  44. 44.
    D. E. McRee, XtalView/Xfit — A versatile program for manipulating atomic coordinates and electron density, J. Struct. Biol. 125(2–5), 156–165 (1999).PubMedCrossRefGoogle Scholar
  45. 45.
    D. E. McRee, Differential evolution for protein crystallographic optimizations, Acta Crystallogr. D Biol. Crystallogr. 60 (Pt 12, No 1), 2276–2279 (2004).PubMedCrossRefGoogle Scholar
  46. 46.
    S. V. Strelkov, and P. Burkhard, Analysis of alpha-helical coiled coils with the program TWISTER reveals a structural mechanism for stutter compensation, J. Struct. Biol. 137(1–2), 54–64 (2002).PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2007

Authors and Affiliations

  • Yasushi Nitanai
    • 1
    • 2
  • Shiho Minakata
    • 1
  • Kayo Maeda
    • 1
  • Naoko Oda
    • 1
  • Yuichiro Maéda
    • 1
    • 2
    • 3
  1. 1.ERATO Actin Filament Dynamics ProjectJSTSayo, HyogoJapan
  2. 2.Laboratory for Structural BiochemistryRIKEN SPring-8 CenterSayo, HyogoJapan
  3. 3.Division of Biological Science, Graduate School of ScienceNagoya UniversityNagoyaJapan

Personalised recommendations