Journal of Biomolecular NMR

, Volume 24, Issue 1, pp 31–39 | Cite as

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

  • Alexander Eletsky
  • Tim Heinz
  • Osvaldo Moreira
  • Alexander Kienhöfer
  • Donald Hilvert
  • Konstantin Pervushin
Article

Abstract

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, 2hJNN and 1hJHN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.

density functional theory hydrogen bonds polarization transfer protein structure scalar couplings TROSY NMR 

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References

  1. Barfield, M., Dingley, A.J., Feigon, J. and Grzesiek, S. (2001) J. Am. Chem. Soc., 123, 4014–4022.Google Scholar
  2. Benedict, H., Shenderovich, I.G., Malkina, O.L., Malkin, V.G., Denisov, G.S., Golubev, N.S. and Limbach, H.H. (2000) J. Am. Chem. Soc., 122, 1979–1988.Google Scholar
  3. Case, D.A., Scheurer, C. and Bruschweiler, R. (2000) J. Am. Chem. Soc., 122, 10390–10397.Google Scholar
  4. Chook, Y.M., Gray, J.V., Ke, H.M. and Lipscomb, W.N. (1994) J. Mol. Biol., 240, 476–500.Google Scholar
  5. Cload, S.T., Liu, D.R., Pastor, R.M. and Schultz, P.G. (1996) J. Am. Chem. Soc., 118, 1787–1788.Google Scholar
  6. Cordier, F. and Grzesiek, S. (1999) J. Am. Chem. Soc., 121, 1601–1602.Google Scholar
  7. Cornilescu, G., Hu, J.S. and Bax, A. (1999a) J. Am. Chem. Soc., 121, 2949–2950.Google Scholar
  8. Cornilescu, G., Ramirez, B.E., Frank, M.K., Clore, G.M., Gronenborn, A.M. and Bax, A. (1999b) J. Am. Chem. Soc., 121, 6275–6279.Google Scholar
  9. de Dios, A.C. and Oldfield, E. (1996) Solid State NMR, 6, 101–125.Google Scholar
  10. Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293–8297.Google Scholar
  11. Dingley, A.J., Cordier, F. and Grzesiek, S. (2001) Concepts Magn. Reson., 13, 103–127.Google Scholar
  12. Dingley, A.J., Masse, J.E., Feigon, J. and Grzesiek, S. (2000) J. Biomol. NMR, 16, 279–289.Google Scholar
  13. Dingley, A.J., Masse, J.E., Peterson, R.D., Barfield, M., Feigon, J. and Grzesiek, S. (1999) J. Am. Chem. Soc., 121, 6019–6027.Google Scholar
  14. Frisch, M.J. et al. (1998) Gaussian98 (Revision A.1), Gaussian, Inc., Pittsburgh PA.Google Scholar
  15. Gray, J.V. and Knowles, J.R. (1994) Biochemistry, 33, 9953–9959.Google Scholar
  16. Gray, J.V., Golinellipimpaneau, B. and Knowles, J.R. (1990a) Biochemistry, 29, 376–383.Google Scholar
  17. Gray, J.V., Eren, D. and Knowles, J.R. (1990b) Biochemistry, 29, 8872–8878.Google Scholar
  18. Guntert, P., Dotsch, V., Wider, G. and Wüthrich, K. (1992) J. Biomol. NMR, 2, 619–629.Google Scholar
  19. Hennig, M. and Geierstanger, B.H. (1999) J. Am. Chem. Soc., 121, 5123–5126.Google Scholar
  20. Kast, P., Hartgerink, J.D., Asif-Ullah, M. and Hilvert, D. (1996) J. Am. Chem. Soc., 118, 3069–3070.Google Scholar
  21. Kutzelnigg, W., Fleischer, U. and Schindler, M. (1991) In NMR: Basic Principles and Progress, Vol. 23, Diehl, P., Fluck, E. and Kosfeld, E. (Eds.), Springer-Verlag, Berlin, pp. 165–262.Google Scholar
  22. Ladner, J.E., Reddy, P., Davis, A., Tordova, M., Howard, A.J. and Gilliland, G.L. (2000) Acta Crystallogr. Sect. D-Biol. Crystallogr., 56, 673–683.Google Scholar
  23. Le, H.B., Pearson, J.G., de Dios, A.C. and Oldfield, E. (1995) J. Am. Chem. Soc., 117, 3800–3807.Google Scholar
  24. Lee, A.Y., Stewart, J.D., Clardy, J. and Ganem, B. (1995) Chem. Biol., 2, 195–203.Google Scholar
  25. Liu, A.Z., Hu, W.D., Majumdar, A., Rosen, M.K. and Patel, D.J. (2000a) J. Biomol. NMR, 17, 79–82.Google Scholar
  26. Liu, A.Z., Hu, W.D., Majumdar, A., Rosen, M.K. and Patel, D.J. (2000b) J. Biomol. NMR, 17, 305–310.Google Scholar
  27. MacBeath, G. and Kast, P. (1998) Biotechniques, 24, 789–794.Google Scholar
  28. Majumdar, A., Kettani, A. and Skripkin, E. (1999) J. Biomol. NMR, 14, 67–70.Google Scholar
  29. Malkin, V.G., Malkina, O.L., Casida, M.E. and Salahub, D.R. (1994) J. Am. Chem. Soc., 116, 5898–5908.Google Scholar
  30. Mattei, P., Kast, P. and Hi1vert, D. (1999) Eur. J. Biochem., 261, 25–32.Google Scholar
  31. Pardi, A., Wagner, G. and Wüthrich, K. (1983) Eur. J. Biochem., 137, 445–454.Google Scholar
  32. Pelton, J.G., Torchia, D.A., Meadow, N.D. and Roseman, S. (1993) Protein Sci., 2, 543–558.Google Scholar
  33. Perdew, J.P. and Wang, Y. (1992) Phys. Rev., B45, 13244–13249.Google Scholar
  34. Pervushin, K., Fernandez, C., Riek, R., Ono, A., Kainosho, M. and Wuthrich, K. (2000) J. Biomol. NMR, 16, 39–46.Google Scholar
  35. Pervushin, K., Ono, A., Fernandez, C., Szyperski, T., Kainosho, M. and Wüthrich, K. (1998) Proc. Natl. Acad. Sci. USA, 95, 14147–14151.Google Scholar
  36. Rajagopalan, J.S., Taylor, K.M. and Jaffe, E.K. (1993) Biochemistry, 32, 3965–3972.Google Scholar
  37. Riek, R., Pervushin, K., Fernandez, C., Kainosho, M. and Wuthrich, K. (2001) J. Am. Chem. Soc., 123, 658–664.Google Scholar
  38. St-Amant, A. and Sa1ahub, D.R. (1990) Chem. Phys. Lett., 169, 387–392.Google Scholar
  39. Wagner, G., Pardi, A. and Wuthrich, K. (1983) J. Am. Chem. Soc., 105, 5948–5949.Google Scholar
  40. Wang, Y.X., Jacob, J., Cordier, F., Wingfield, P., Stahl, S.J., Lee-Huang, S., Torchia, D., Grzesiek, S. and Bax, A. (1999) J. Biomol. NMR, 14, 181–184.Google Scholar
  41. Wohnert, J., Dingley, A.J., Stoldt, M., Gorlach, M., Grzesiek, S. and Brown, L.R. (1999) Nucl. Acids Res., 27, 3104–3110.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Alexander Eletsky
    • 1
  • Tim Heinz
    • 1
  • Osvaldo Moreira
    • 1
  • Alexander Kienhöfer
    • 2
  • Donald Hilvert
    • 2
  • Konstantin Pervushin
    • 1
  1. 1.Laboratorium für Physikalische ChemieSwiss Federal Institute of Technology, ETH HönggerbergZürichSwitzerland
  2. 2.Laboratorium für Organische ChemieSwiss Federal Institute of Technology, ETH HönggerbergZürichSwitzerland

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