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A Case History

NMR Studies of the Structure of a Small Protein, ω-Conotoxin MVIIA

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Protein NMR Techniques

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 60))

Abstract

ω-Conotoxin MVIIA is a small toxin protein derived from the venom of a fish-hunting sea snail Conus mugus. The cone snail venom proteins, in general, display potency and selectivity toward certain pharmacological receptors (1), which has made them objects of intense study. The three-dimensional (3D) conformations of these molecules, in particular, have excited interest as possibly providing structural leads in rational drug design programs. The conotoxins are highly constrained conformationally by two or three disulfide bonds; these disulfide crosslinking motifs are strongly conserved, so it is thought that the pharmacological specificity of the various congeners resides in the high degree of variability manifested in the loop regions between the bridging cysteine residues (2). To date, there have been no X-ray crystal structures reported for a conotoxin, although NMR structures have been reported for α-conotoxin GI (3,4), µ-conotoxin GIIIA (5,6), and ω-conotoxin GVIA (7–10); also for ω-conotoxins MVIIA (11) and MVIIC (12,13). Of these, the structures of µ-conotoxin GIIIA (14), ω-conotoxin GVIA (8,10), ω-conotoxin MVIIA (11) and ω-conotoxin MVIIC (13) have been deposited in the Protein Data Bank at Brookhaven National Laboratory (15,16).

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References

  1. Gray, W. R., Olivera, B. M., and Cruz, L. J. (1988) Peptide toxins from venomous Conus snails. Ann. Rev. Biochem. 57, 665–700.

    Article  PubMed  CAS  Google Scholar 

  2. Olivera, B. M., Rivier, J., Scott, J. K., Hillyard, D. R., and Cruz, L. J. (1991) Conotoxins. J. Biol. Chem. 266, 22,067–22,070.

    PubMed  CAS  Google Scholar 

  3. Kobayashi, Y., Ohkubo, T., Kyogoku, Y., Nishiuchi, Y., Sakakibara, S., Braun, W., and Go, N. (1989) Solution conformation of conotoxin GI determined by 1H nuclear magnetic resonance spectroscopy and distance geometry calculations. Biochemistry 28, 4853–4860.

    Article  PubMed  CAS  Google Scholar 

  4. Pardi, A., Galdes, A., Florance, J., and Maniconte, D. (1989) Solution structures of α-conotoxin GI determined by two-dimensional NMR spectroscopy. Biochemistry 28, 5494–5501.

    Article  PubMed  CAS  Google Scholar 

  5. Lancelin, J.-M., Kohda, D., Tate, S., Yanagawa, Y., Abe, T., Satake, M., and Inagaki, F. (1991) Tertiary structure of conotoxin GIIIA in aqueous solution. Biochemistry 30, 6908–6916.

    Article  PubMed  CAS  Google Scholar 

  6. Ott, K.-H., Becker, S., Gordon, R. D., and Ruterjans, H. (1991) Solution structure of µ-conotoxin GIIIA analysed by 2D NMR and distance geometry calculations. FEBS Lett. 278, 160–166.

    Article  PubMed  CAS  Google Scholar 

  7. Sevilla, P., Briux, M., Santoro, J., Gago, F., Garcia, A. G., and Rico, M. (1993) Three-dimensional structure of ω-conotoxin GVIA determined by 1H NMR. Biochem. Biophys. Res. Commun. 192, 1238–1244.

    Article  PubMed  CAS  Google Scholar 

  8. Davis, J. H., Bradley, E. K., Miljanich, G. P., Nadasdi, L., Ramachandran, J., and Basus, V. J. (1993) Solution structure of ω-conotoxin GVIA using 2-D NMR spectroscopy and relaxation matrix analysis. Biochemistry 32, 7396–7405.

    Article  PubMed  CAS  Google Scholar 

  9. Skalicky, J. J., Metzler, W. J., Ciesla, D. J., Galdes, A., and Pardi, A. (1993) Solution structure of the calcium channel antagonist ω-conotoxin GVIA. Protein Sci. 2, 1591–1603.

    Article  PubMed  CAS  Google Scholar 

  10. Pallaghy, P. K., Duggan, B. M., Pennington, M. W., and Norton, R. S. (1993) Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin. J. Mol. Biol. 234(2), 405–420.

    Article  PubMed  CAS  Google Scholar 

  11. Kohno, T., Kim, J.-I., Kobayashi, K., Kodera, Y., Maeda, T., and Sato, K. (1995) Three-dimensional structure in solution of the calcium channel blocker ω-conotoxin MVIIA. Biochemistry 34, 10,256–10,265.

    Article  PubMed  CAS  Google Scholar 

  12. Nemoto, N., Kubo, S., Yoshida, T., Chino, N., Kimura, T., Sakakibara, S., Kyogoku, Y., and Kobayashi, Y. (1995) Solution structure of ω-conotoxin MVIIC determined by NMR. Biochem. Biophys. Res. Commun. 207(2), 695–700.

    Article  PubMed  CAS  Google Scholar 

  13. Farr-Jones, S., Miljanich, G. P., Nadasdi, L., Ramachandran, J., and Basus, V. (1995) Solution structure of ω-conotoxin MVIIC, a high affinity ligand of P-type calcium channels, using 1H NMR spectroscopy and complete relaxation matrix analysis. J. Mol. Biol. 248, 106–124.

    Article  PubMed  CAS  Google Scholar 

  14. Wakamatsu, K., Kohda, D., Hatanaka, H., Lancelin, J.-M., Ishida, Y., Ohya, M., Nakamura, H., Inagaki, F., and Sato, K. (1992) Structure—activity relationships of µ-conotoxin GIIIA structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations. Biochemistry 31, 12,577–12,584.

    Article  PubMed  CAS  Google Scholar 

  15. Bernstein, F. C., Koetzle, T. F., Willlams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) The Protein Data Bank a computer-based archival file for macromolecular structure. J. Mol. Biol. 112, 535–542.

    Article  PubMed  CAS  Google Scholar 

  16. Abola, E. E., Bernstein, F. C., Bryant, S. H., Koetzle, T. F., and Weng, J. (1987) in Crystallographic Databases—Information Content, Software Systems, Scientific Applications. (Allen, F. H., Bergerhoff, G., and Sievers, R., eds.) Data Commission of the International Union of Crystallography, Bonn, Germany, pp. 107–132.

    Google Scholar 

  17. LeMaster, D. M. (1994) Isotope labelling in solution protein assignment and structural analysis. Prog. NMR Spectrosc. 26, 371–419.

    Article  CAS  Google Scholar 

  18. Wagner, G. (1993) Prospects for NMR of large proteins. J. Biomol. NMR 3, 375–385.

    Article  PubMed  CAS  Google Scholar 

  19. Clore, G. M. and Gronenborn, A. M. (1991) Two-, three-, and four-dimensional NMR methods for obtaining larger and more precise three-dimensional structures of proteins in solution. Annu. Rev. Biophys. Biophys. Chem. 20, 29–63.

    Article  PubMed  CAS  Google Scholar 

  20. Peng, J. W. and Wagner, G. (1994) Investigation of protein motions via relaxation measurements, in Methods in Enzymology Nuclear Magnetic Resonance Part C (James, T. L. and Oppenheimer, N. J., eds.) Academic, San Diego, CA, pp. 563–596.

    Chapter  Google Scholar 

  21. Rance, M., Sorensen, O. W., Bodenhausen, G., Wagner, G., Ernst, R. R., and Wuthrich, K. (1983) Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun. 117(2), 479–485.

    Article  PubMed  CAS  Google Scholar 

  22. Davis, D. G. and Bax, A. (1985) Assignment of complex 1H NMR spectra via 2D homonuclear Hartmann-Hahn spectroscopy. J. Am. Chem. Soc. 107, 2820,2821.

    Article  CAS  Google Scholar 

  23. Mueller, L. (1987) P.E.COSY, a simple alternative to E. COSY. J. Magn. Reson. 72, 191–196.

    CAS  Google Scholar 

  24. Wuethrich, K. (1986) NMR of Proteins and Nucleic Acids Wiley, New York.

    Google Scholar 

  25. Redfield, C. and Dobson, C. M. (1988) Sequential 1H NMR assignments and secondary structure of hen egg white lysozyme in solution. Biochemistry 27, 122–136.

    Article  PubMed  CAS  Google Scholar 

  26. Englander, S. W. and Kallenbach, N. R. (1983) Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys. 16, 521–655.

    Article  PubMed  CAS  Google Scholar 

  27. Dyson, H. J., Rance, M., Houghten, R. A., Lerner, R. A., and Wright, P. E. (1988) Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn. J. Mol. Biol. 201, 161–200.

    Article  PubMed  CAS  Google Scholar 

  28. Dyson, H. J. and Wright, P. E. (1991). Defining solution conformations of small linear peptides. Annu. Rev. Biophys. Biophys. Chem. 20, 519–538.

    Article  PubMed  CAS  Google Scholar 

  29. Barsukov, I. L. and Lian, L.-Y. (1993) Structure determination from NMR data I, in NMR of Macromolecules: A Practical Approach (Roberts, G. C. K., ed.) Oxford University Press, Oxford, UK, pp. 315–357.

    Google Scholar 

  30. Wishart, D. S. and Sykes, B. D. (1994) Chemical shifts as a tool for structure determination, in Methods in Enzymology Vol. 239 Nuclear Magnetic Resonance Part C. (James, T. L. and Oppenheimer, N. J., eds.) Academic, San Diego, pp. 363–392.

    Chapter  Google Scholar 

  31. Lee, J. P., Stimson, E. R., Ghilardt, J. R., Mantyh, P. W., Lu, Y.-A., Felix, A. M., Llanos, W., Behbin, A., Cummings, M., van Criekinge, M., Timms, W., and Maggio, J. E. (1995) 1H NMR of Aβ amyloid peptide congeners in water solution. Conformational changes correlate with plaque competence. Biochemistry 34, 5191–5200.

    Article  PubMed  CAS  Google Scholar 

  32. Narasimhan, L., Singh, J., Humble, C., Guruprasad, K., and Blundell, T. L. (1994). Snail and spider toxins share a similar tertiary structure and “cystine motif”. Nat. Structur. Biol. 1, 850–852.

    Article  CAS  Google Scholar 

  33. Miljanich, G. P., Bitner, R. S., Bowersox, S. S., Fox, J. A., Valentino, K. L., and Yamashiro, D. H. Method of treating ischaemia-related neuronal damage. United States Patent number 5051403, September 24, 1991.

    Google Scholar 

  34. Kim, J. I., Takahashi, M., Ohtake, A., Wakamiya, A., and Sato, K. (1995) Tyr13 is essential for the activity of ω-conotoxin MVIIA and GVIA, specific N-type calcium channel blockers. Biochem Biophys. Res. Commun. 206, 449–454.

    Article  PubMed  CAS  Google Scholar 

  35. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) Investigation of exchange processes by 2D NMR spectroscopy. J. Chem. Phys. 71(11), 4546–4553.

    Article  CAS  Google Scholar 

  36. Marion, D., Ikura, M., Tschudin, R., and Bax, A. (1989) Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins. J. Magn. Reson. 85, 393–399.

    CAS  Google Scholar 

  37. Shaka, A. J., Lee, C. J., and Pines, A. (1988) Iterative schemes for bilinear operators; application to spin decoupling. J. Magn. Reson. 77, 274–293.

    Google Scholar 

  38. Marion, D., Ikura, M., and Bax, A. (1989) Improved solvent suppression in 1-and 2D NMR spectra by convolution of time-domain data. J. Magn. Reson. 84, 425–430.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Analytical Sciences Department, SmithKline Beecham Pharmaceuticals, Welwyn, Hertfordshire, UK

    Lesley K. MacLachlan, David A. Middleton, Andrew J. Edwards & David G. Reid

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  1. Lesley K. MacLachlan
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  2. David A. Middleton
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  3. Andrew J. Edwards
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  1. SmithKline Beecham Pharmaceuticals, Welwyn, Hertfordshire, UK

    David G. Reid

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© 1997 Humana Press Inc.

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MacLachlan, L.K., Middleton, D.A., Edwards, A.J., Reid, D.G. (1997). A Case History. In: Reid, D.G. (eds) Protein NMR Techniques. Methods in Molecular Biology™, vol 60. Humana Press. https://doi.org/10.1385/0-89603-309-0:337

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  • DOI: https://doi.org/10.1385/0-89603-309-0:337

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-309-2

  • Online ISBN: 978-1-59259-546-4

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