The Conformation of a Signal Peptide Bound by Escherichia coli Preprotein Translocase SecA (original) (raw)

Abstract

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (K d ϳ 10 ؊5 M). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an ␣-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.

Loading...

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (59)

  1. Gierasch, L. M. (1989) Biochemistry 28, 923-930
  2. von Heijne, G. (1985) J. Mol. Biol. 184, 99 -105
  3. Gennity, J., Goldstein, J., and Inouye, M. (1990) J. Bioenerg. Biomembr. 22, 233-269
  4. Izard, J. W., and Kendall, D. A. (1994) Mol. Microbiol. 13, 765-773
  5. Izard, J. W., Rusch, S. L., and Kendall, D. A. (1996) J. Biol. Chem. 271, 21579 -21582
  6. Fekkes, P., and Driessen, A. J. (1999) Microbiol. Mol. Biol. Rev. 63, 161-173
  7. Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280
  8. Vrontou, E., and Economou, A. (2004) Biochim. Biophys. Acta 1694, 67-80
  9. Hartl, F. U., Lecker, S., Schiebel, E., Hendrick, J. P., and Wickner, W. (1990) Cell 63, 269 -279
  10. Economou, A., and Wickner, W. (1994) Cell 78, 835-843
  11. Schiebel, E., Driessen, A. J., Hartl, F. U., and Wickner, W. (1991) Cell 64, 927-939
  12. van der Wolk, J. P., de Wit, J. G., and Driessen, A. J. (1997) EMBO J. 16, 7297-7304
  13. Dalbey, R., and Chen, M. (2004) Biochim. Biophys. Acta 1694, 37-53
  14. Derman, A. I., Puziss, J. W., Bassford, P. J., and Beckwith, J. (1993) EMBO J. 12, 879 -888
  15. Emr, S. D., Hanley-Way, S., and Silhavy, T. J. (1981) Cell 23, 79 -88
  16. Flower, A. M., Doebele, R. C., and Silhavy, T. J. (1994) J. Bacteriol. 176, 5607-5614
  17. Stader, J., Benson, S. A., and Silhavy, T. J. (1986) J. Biol. Chem. 261, 15075-15080
  18. Bieker, K. L., and Silhavy, T. J. (1990) Cell 61, 833-842
  19. Veenendaal, A. K., van der Does, C., and Driessen, A. J. (2004) Biochim. Biophys. Acta 1694, 81-95
  20. van der Wolk, J. P., Fekkes, P., Boorsma, A., Huie, J. L., Silhavy, T. J., and Driessen, A. J. (1998) EMBO J. 17, 3631-3639
  21. Duong, F., and Wickner, W. (1999) EMBO J. 18, 3263-3270
  22. Schmidt, M., Ding, H., Ramamurthy, V., Mukerji, I., and Oliver, D. (2000) J. Biol. Chem. 275, 15440 -15448
  23. Triplett, T. L., Sgrignoli, A. R., Gao, F., Yang, Y., Tai, P. C., and Gierasch, L. M. (2001) J. Biol. Chem. 276, 19648 -19655
  24. Miller, A., Wang, L., and Kendall, D. A. (1998) J. Biol. Chem. 273, 11409 -11412
  25. Kimura, E., Akita, M., Matsuyama, S., and Mizushima, S. (1991) J. Biol. Chem. 266, 6600 -6606
  26. Wang, L., Miller, A., and Kendall, D. A. (2000) J. Biol. Chem. 275, 10154 -10159
  27. Baud, C., Karamanou, S., Sianidis, G., Vrontou, E., Politou, A. S., and Economou, A. (2002) J. Biol. Chem. 277, 13724 -13731
  28. Cunningham, K., and Wickner, W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8630 -8634
  29. Benach, J., Chou, Y.-T., Fak, J. J., Itkin, A., Nicolae, D. D., Smith, P. C., Wittrock, G., Floyd, D., Gierasch, L. M., and Hunt, J. F. (2003) J. Biol. Chem. 278, 3628 -3638
  30. Or, E., Navon, A., and Rapoport, T. (2002) EMBO J. 21, 4470 -4479
  31. Briggs, M. S., Cornell, D. G., Dluhy, R. A., and Gierasch, L. M. (1986) Science 233, 206 -208
  32. Bruch, M. D., McKnight, C. J., and Gierasch, L. M. (1989) Biochemistry 28, 8554 -8561
  33. McKnight, C. J., Briggs, M. S., and Gierasch, L. M. (1989) J. Biol. Chem. 264, 17293-17297
  34. Bruch, M. D., and Gierasch, L. M. (1990) J. Biol. Chem. 265, 3851-3858
  35. Rizo, J., Blanco, F. J., Kobe, B., Bruch, M. D., and Gierasch, L. M. (1993) Biochemistry 32, 4881-4894
  36. Wang, Z. L., Jones, J. D., Rizo, J., and Gierasch, L. M. (1993) Biochemistry 32, 13991-13999
  37. Emr, S. D., and Silhavy, T. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4599 -4603
  38. Osborne, A. R., Clemons, W. M., Jr., and Rapoport, T. A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 10937-10942
  39. Sharma, V., Arockiasamy, A., Ronning, D. R., Savva, C. G., Holzenburg, A., Braun- stein, M., Jacobs, W. R., Jr., and Sacchettini, J. C. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2243-2248
  40. Hunt, J. F., Weinkauf, S., Henry, L., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018 -2026
  41. Baud, C., Papanikou, E., Karamanou, S., Sianidis, G., Kuhn, A., and Economou, A. (2005) Protein Expression Purif. 40, 336 -339
  42. Kourtz, L., and Oliver, D. (2000) Mol. Microbiol. 37, 1342-1356
  43. Ulbrandt, N. D., London, E., and Oliver, D. B. (1992) J. Biol. Chem. 267, 15184 -15192
  44. Breukink, E., Nouwen, N., van Raalte, A., Mizushima, S., Tommassen, J., and de Kruijff, B. (1995) J. Biol. Chem. 270, 7902-7907
  45. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992) Nature 355, 455-457
  46. Wang, Z., Feng, H.-P., Landry, S. J., Maxwell, J., and Gierasch, L. M. (1999) Biochem- istry 38, 12537-12546
  47. Cavanagh, J., Fairbrother, W. J., Palmer, A. G., III, and Skelton, N. J. (1996) Protein NMR Spectroscopy: Principles and Practice, 1st Ed., pp. 95-243, Academic Press, San Diego, CA
  48. Wu ¨thrich, K. (1986) NMR of Proteins and Nucleic Acids, pp. 27-175, John Wiley and Sons, New York
  49. Lian, L. Y., Barsukov, I. L., Sutcliffe, M. J., Sze, K. H., and Roberts, G. C. (1994) Methods Enzymol. 239, 657-700
  50. Clore, G. M., and Gronenborn, A. M. (1982) J. Mag. Res. 48, 402-417
  51. Clore, G. M., and Gronenborn, A. M. (1983) J. Mag. Res. 53, 423-442
  52. Campbell, A. P., and Sykes, B. D. (1993) Ann. Rev. Biophys. Biomol. Struct. 22, 99 -122
  53. Briggs, M. S., and Gierasch, L. M. (1984) Biochemistry 23, 3111-3114
  54. McKnight, C. J., Stradley, S. J., Jones, J. D., and Gierasch, L. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5799 -5803
  55. Hoyt, D. W., and Gierasch, L. M. (1991) J. Biol. Chem. 266, 14406 -14412
  56. Goldstein, J., Lehnhardt, S., and Inouye, M. (1991) J. Biol. Chem. 266, 14413-14417
  57. Chou, Y. T., Swain, J. F., and Gierasch, L. M. (2002) J. Biol. Chem. 277, 50985-50990
  58. Sianidis, G., Karamanou, S., Vrontou, E., Boulias, K., Repanas, K., Kyrpides, N., Poli- tou, A. S., and Economou, A. (2001) EMBO J. 20, 961-970
  59. Mori, H., Araki, M., Hikita, C., Tagaya, M., and Mizushima, S. (1997) Biochim. Bio- phys. Acta 1326, 23-36