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The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein

Abstract

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ¿1H¿-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ¿1H¿-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.

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Selected References

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  1. Cho H. S., Liu C. W., Damberger F. F., Pelton J. G., Nelson H. C., Wemmer D. E. Yeast heat shock transcription factor N-terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy. Protein Sci. 1996 Feb;5(2):262–269. doi: 10.1002/pro.5560050210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Daughdrill G. W., Chadsey M. S., Karlinsey J. E., Hughes K. T., Dahlquist F. W. The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, sigma 28. Nat Struct Biol. 1997 Apr;4(4):285–291. doi: 10.1038/nsb0497-285. [DOI] [PubMed] [Google Scholar]
  3. Farrow N. A., Muhandiram R., Singer A. U., Pascal S. M., Kay C. M., Gish G., Shoelson S. E., Pawson T., Forman-Kay J. D., Kay L. E. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994 May 17;33(19):5984–6003. doi: 10.1021/bi00185a040. [DOI] [PubMed] [Google Scholar]
  4. Fletcher C. M., McGuire A. M., Gingras A. C., Li H., Matsuo H., Sonenberg N., Wagner G. 4E binding proteins inhibit the translation factor eIF4E without folded structure. Biochemistry. 1998 Jan 6;37(1):9–15. doi: 10.1021/bi972494r. [DOI] [PubMed] [Google Scholar]
  5. Mader S., Lee H., Pause A., Sonenberg N. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol Cell Biol. 1995 Sep;15(9):4990–4997. doi: 10.1128/mcb.15.9.4990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Matsuo H., Li H., Wagner G. A sensitive HN(CA)CO experiment for deuterated proteins. J Magn Reson B. 1996 Jan;110(1):112–115. doi: 10.1006/jmrb.1996.0018. [DOI] [PubMed] [Google Scholar]
  7. Pause A., Belsham G. J., Gingras A. C., Donzé O., Lin T. A., Lawrence J. C., Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994 Oct 27;371(6500):762–767. doi: 10.1038/371762a0. [DOI] [PubMed] [Google Scholar]
  8. Penkett C. J., Redfield C., Dodd I., Hubbard J., McBay D. L., Mossakowska D. E., Smith R. A., Dobson C. M., Smith L. J. NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein. J Mol Biol. 1997 Nov 28;274(2):152–159. doi: 10.1006/jmbi.1997.1369. [DOI] [PubMed] [Google Scholar]
  9. Plaxco K. W., Gross M. Cell biology. The importance of being unfolded. Nature. 1997 Apr 17;386(6626):657–659. doi: 10.1038/386657a0. [DOI] [PubMed] [Google Scholar]
  10. Rousseau D., Gingras A. C., Pause A., Sonenberg N. The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth. Oncogene. 1996 Dec 5;13(11):2415–2420. [PubMed] [Google Scholar]
  11. Talluri S., Wagner G. An optimized 3D NOESY-HSQC. J Magn Reson B. 1996 Aug;112(2):200–205. doi: 10.1006/jmrb.1996.0132. [DOI] [PubMed] [Google Scholar]
  12. Thomas G., Hall M. N. TOR signalling and control of cell growth. Curr Opin Cell Biol. 1997 Dec;9(6):782–787. doi: 10.1016/s0955-0674(97)80078-6. [DOI] [PubMed] [Google Scholar]
  13. Uesugi M., Nyanguile O., Lu H., Levine A. J., Verdine G. L. Induced alpha helix in the VP16 activation domain upon binding to a human TAF. Science. 1997 Aug 29;277(5330):1310–1313. doi: 10.1126/science.277.5330.1310. [DOI] [PubMed] [Google Scholar]
  14. Wishart D. S., Bigam C. G., Holm A., Hodges R. S., Sykes B. D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR. 1995 Jan;5(1):67–81. doi: 10.1007/BF00227471. [DOI] [PubMed] [Google Scholar]
  15. Zhang O., Kay L. E., Olivier J. P., Forman-Kay J. D. Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR. 1994 Nov;4(6):845–858. doi: 10.1007/BF00398413. [DOI] [PubMed] [Google Scholar]