Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1

Nature Structural & Molecular Biology volume 17, pages 1367–1376 (2010)Cite this article

Subjects

Abstract

Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP−CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Enhancement of the PKI NES–CRM1 interaction by Φ0Leu and the SPN1 fusion module.
Figure 2: Crystal structure of a classic PKI-type NES bound to CRM1.
Figure 3: Solution NMR structure of the CRM1-bound PKI Φ0Leu NES.
Figure 4: Φ preferences in PKI-type NESs.
Figure 5: Supraphysiological and attenuated NESs.
Figure 6: Export activity of NESs with unconventionally spaced Φ residues.
Figure 7: Structure of the CRM1-bound HIV-1 Rev NES.
Figure 8: Redefinition of the NES consensus.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Görlich, D. & Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 (1999).

    Article  Google Scholar 

  2. Cook, A.G. & Conti, E. Nuclear export complexes in the frame. Curr. Opin. Struct. Biol. 20, 247–252 (2010).

    Article  CAS  Google Scholar 

  3. Adachi, Y. & Yanagida, M. Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery. J. Cell Biol. 108, 1195–1207 (1989).

    Article  CAS  Google Scholar 

  4. Fornerod, M., Ohno, M., Yoshida, M. & Mattaj, I.W. Crm1 is an export receptor for leucine rich nuclear export signals. Cell 90, 1051–1060 (1997).

    Article  CAS  Google Scholar 

  5. Stade, K., Ford, C.S., Guthrie, C. & Weis, K. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90, 1041–1050 (1997).

    Article  CAS  Google Scholar 

  6. Nishi, K. et al. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol. Chem. 269, 6320–6324 (1994).

    CAS  PubMed  Google Scholar 

  7. Wolff, B., Sanglier, J.J. & Wang, Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139–147 (1997).

    Article  CAS  Google Scholar 

  8. Kutay, U., Bischoff, F.R., Kostka, S., Kraft, R. & Görlich, D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell 90, 1061–1071 (1997).

    Article  CAS  Google Scholar 

  9. Matsuura, Y. & Stewart, M. Structural basis for the assembly of a nuclear export complex. Nature 432, 872–877 (2004).

    Article  CAS  Google Scholar 

  10. Cook, A.G., Fukuhara, N., Jinek, M. & Conti, E. Structures of the tRNA export factor in the nuclear and cytosolic states. Nature 461, 60–65 (2009).

    Article  CAS  Google Scholar 

  11. Okada, C. et al. A high-resolution structure of the pre-microRNA nuclear export machinery. Science 326, 1275–1279 (2009).

    Article  CAS  Google Scholar 

  12. Monecke, T. et al. Crystal Structure of the Nuclear Export Receptor CRM1 in Complex with Snurportin1 and RanGTP. Science 324, 1087–1091 (2009).

    Article  CAS  Google Scholar 

  13. Dong, X. et al. Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 458, 1136–1141 (2009).

    Article  CAS  Google Scholar 

  14. Koyama, M. & Matsuura, Y. An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1. EMBO J. 29, 2002–2013 (2010).

    Article  CAS  Google Scholar 

  15. Ho, J.H., Kallstrom, G. & Johnson, A.W. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151, 1057–1066 (2000).

    Article  CAS  Google Scholar 

  16. Gadal, O. et al. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol. Cell. Biol. 21, 3405–3415 (2001).

    Article  CAS  Google Scholar 

  17. Moy, T.I. & Silver, P.A. Requirements for the nuclear export of the small ribosomal subunit. J. Cell Sci. 115, 2985–2995 (2002).

    CAS  PubMed  Google Scholar 

  18. Thomas, F. & Kutay, U. Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway. J. Cell Sci. 116, 2409–2419 (2003).

    Article  CAS  Google Scholar 

  19. Malim, M.H., McCarn, D.F., Tiley, L.S. & Cullen, B.R. Mutational definition of the human immunodeficiency virus type 1 Rev activation domain. J. Virol. 65, 4248–4254 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W. & Lührmann, R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475–483 (1995).

    Article  CAS  Google Scholar 

  21. Wen, W., Meinkoth, J., Tsien, R. & Taylor, S. Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473 (1995).

    Article  CAS  Google Scholar 

  22. Bohnsack, M.T. et al. Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 21, 6205–6215 (2002).

    Article  CAS  Google Scholar 

  23. Richards, S.A., Lounsbury, K.M., Carey, K.L. & Macara, I.G. A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1. J. Cell Biol. 134, 1157–1168 (1996).

    Article  CAS  Google Scholar 

  24. Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I.W. & Görlich, D. The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 16, 6535–6547 (1997).

    Article  CAS  Google Scholar 

  25. Kutay, U. & Guttinger, S. Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol. 15, 121–124 (2005).

    Article  CAS  Google Scholar 

  26. Bogerd, H.P., Fridell, R.A., Benson, R.E., Hua, J. & Cullen, B.R. Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay. Mol. Cell. Biol. 16, 4207–4214 (1996).

    Article  CAS  Google Scholar 

  27. Zhang, M.J. & Dayton, A.I. Tolerance of diverse amino acid substitutions at conserved positions in the nuclear export signal (NES) of HIV-1 Rev. Biochem. Biophys. Res. Commun. 243, 113–116 (1998).

    Article  CAS  Google Scholar 

  28. Kosugi, S., Hasebe, M., Tomita, M. & Yanagawa, H. Nuclear export signal consensus sequences defined using a localization-based yeast selection system. Traffic 9, 2053–2062 (2008).

    Article  CAS  Google Scholar 

  29. Huber, J. et al. Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure. EMBO J. 17, 4114–4126 (1998).

    Article  CAS  Google Scholar 

  30. Paraskeva, E. et al. CRM1-mediated recycling of snurportin 1 to the cytoplasm. J. Cell Biol. 145, 255–264 (1999).

    Article  CAS  Google Scholar 

  31. Engelsma, D., Bernad, R., Calafat, J. & Fornerod, M. Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358. EMBO J. 23, 3643–3652 (2004).

    Article  CAS  Google Scholar 

  32. Engelsma, D. et al. A supraphysiological nuclear export signal is required for parvovirus nuclear export. Mol. Biol. Cell 19, 2544–2552 (2008).

    Article  CAS  Google Scholar 

  33. Cook, A., Bono, F., Jinek, M. & Conti, E. Structural biology of nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647–671 (2007).

    Article  CAS  Google Scholar 

  34. Rice, L.M. & Brunger, A.T. Torsion angle dynamics: reduced variable conformational sampling enhances crystallographic structure refinement. Proteins 19, 277–290 (1994).

    Article  CAS  Google Scholar 

  35. Kudo, N. et al. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96, 9112–9117 (1999).

    Article  CAS  Google Scholar 

  36. Nilges, M. A calculation strategy for the structure determination of symmetrical dimers by H-1-NMR. Proteins 17, 297–309 (1993).

    Article  CAS  Google Scholar 

  37. Nilges, M. & O'Donoghue, S.I. Ambiguous NOEs and automated NOE assignment. Prog. Nucl. Magn. Reson. Spectrosc. 32, 107–139 (1998).

    Article  CAS  Google Scholar 

  38. Madl, T., Bermel, W. & Zangger, K. Use of relaxation enhancements in a paramagnetic environment for the structure determination of proteins using NMR spectroscopy. Angew. Chem. Int. Ed. 48, 8259–8262 (2009).

    Article  CAS  Google Scholar 

  39. Askjaer, P. et al. RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. Mol. Cell. Biol. 19, 6276–6285 (1999).

    Article  CAS  Google Scholar 

  40. Hantschel, O. et al. Structural basis for the cytoskeletal association of Bcr-Abl/c-Abl. Mol. Cell 19, 461–473 (2005).

    Article  CAS  Google Scholar 

  41. Taagepera, S. et al. Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase. Proc. Natl. Acad. Sci. USA 95, 7457–7462 (1998).

    Article  CAS  Google Scholar 

  42. Hutchinson, E.G. & Thornton, J.M. PROMOTIF—a program to identify and analyze structural motifs in proteins. Protein Sci. 5, 212–220 (1996).

    Article  CAS  Google Scholar 

  43. Ribbeck, K. & Görlich, D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21, 2664–2671 (2002).

    Article  CAS  Google Scholar 

  44. Weiss, M.S. & Hilgenfeld, R. On the use of the merging R factor as a quality indicator for X-ray data. J. Appl. Cryst. 30, 203–205 (1997).

    Article  CAS  Google Scholar 

  45. Linge, J.P., Williams, M.A., Spronk, C.A., Bonvin, A.M. & Nilges, M. Refinement of protein structures in explicit solvent. Proteins 50, 496–506 (2003).

    Article  CAS  Google Scholar 

  46. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  Google Scholar 

  47. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  Google Scholar 

  48. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  49. Brunger, A.T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

  50. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  Google Scholar 

  51. McCoy, A.J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D Biol. Crystallogr. 63, 32–41 (2007).

    Article  CAS  Google Scholar 

  52. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  53. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  54. Riek, R., Wider, G., Pervushin, K. & Wüthrich, K. Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc. Natl. Acad. Sci. USA 96, 4918–4923 (1999).

    Article  CAS  Google Scholar 

  55. Tugarinov, V., Hwang, P.M., Ollerenshaw, J.E. & Kay, L.E. Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J. Am. Chem. Soc. 125, 10420–10428 (2003).

    Article  CAS  Google Scholar 

  56. Bermel, W., Bertini, I., Felli, I.C., Piccioli, M. & Pierattelli, R. 13C-detected protonless NMR spectroscopy of proteins in solution. Prog. Nucl. Magn. Reson. Spectrosc. 48, 25–45 (2006).

    Article  CAS  Google Scholar 

  57. Oh, B.H., Westler, W.M., Darba, P. & Markley, J.L. Protein C-13 spin systems by a single two-dimensional nuclear magnetic-resonance experiment. Science 240, 908–911 (1988).

    Article  CAS  Google Scholar 

  58. Delaglio, F. et al. Nmrpipe—a multidimensional spectral processing system based on unix pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

  59. Johnson, B.A. & Blevins, R.A. NMRView: a computer-program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  Google Scholar 

  60. Pintacuda, G. & Otting, G. Identification of protein surfaces by NMR measurements with a paramagnetic Gd(III) chelate. J. Am. Chem. Soc. 124, 372–373 (2002).

    Article  CAS  Google Scholar 

  61. Linge, J.P., Habeck, M., Rieping, W. & Nilges, M. ARIA: automated NOE assignment and NMR structure calculation. Bioinformatics 19, 315–316 (2003).

    Article  CAS  Google Scholar 

  62. Linge, J.P., O'Donoghue, S.I. & Nilges, M. Automated assignment of ambiguous nuclear overhauser effects with ARIA. Methods Enzymol. 339, 71–90 (2001).

    Article  CAS  Google Scholar 

  63. Adam, S.A., Marr, R.S. & Gerace, L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816 (1990).

    Article  CAS  Google Scholar 

  64. Stüven, T., Hartmann, E. & Görlich, D. Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes. EMBO J. 22, 5928–5940 (2003).

    Article  Google Scholar 

  65. Leno, G.H. & Laskey, R.A. DNA replication in cell-free extracts from Xenopus laevis. Methods Cell Biol. 36, 561–579 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Berndt and A. Dickmanns for performing the final purification step and the crystallization of the RanGTP–CRM1 complex, T.A. Rapoport, S. Frey, S. Güttler, C. Siebenhaar and B. Hülsmann for critical reading of the manuscript, B. Hülsmann for preparing the Xenopus egg extract, the Bavarian NMR Centre (BNMRZ) for NMR measurement time, the staff of synchrotron beamlines at the Swiss Light Source (SLS, Villigen, X10SA, PXII) and the European Synchrotron Radiation Facility (ESRF, Grenoble, ID 23-2) for assistance during data collection, and M. Wahl, V. Pena and J. Wawrzinek for setting up the crystallization facility at the MPI. This study was funded by the Max-Planck-Gesellschaft, the Boehringer Ingelheim Fonds and the Alfried Krupp von Bohlen und Halbach Foundation (fellowships to T.G.), EMBO (fellowship to T. Madl), the Austrian Science Fund (FWF, Schrödinger fellowship to T. Madl), European Commission contract 3D Repertoire (LSHG-CT-2005-512028 to M.S.) and contract EU-NMR (No. RII3-026145).

Author information

Author notes

  1. Thomas Güttler, Tobias Madl and Piotr Neumann: These authors contributed equally to this work.

Authors and Affiliations

  1. Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany

    Thomas Güttler, Danilo Deichsel & Dirk Görlich

  2. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany

    Tobias Madl, Lorenzo Corsini & Michael Sattler

  3. Munich Center for Integrated Protein Science at Department Chemie, Technische Universität München, Garching, Germany

    Tobias Madl, Lorenzo Corsini & Michael Sattler

  4. Abteilung für Molekulare Strukturbiologie, Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Georg-August-Universität Göttingen, Göttingen, Germany

    Piotr Neumann, Thomas Monecke & Ralf Ficner

Authors
  1. Thomas Güttler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tobias Madl
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Piotr Neumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danilo Deichsel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lorenzo Corsini
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas Monecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ralf Ficner
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael Sattler
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dirk Görlich
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.G. conceived biochemical and crystallization experiments, prepared protein constructs (with the help of D.D.), performed the biochemical experiments and export assays, crystallized the export complexes, contributed to crystallographic data collection and structure refinement, prepared the samples for the NMR study, interpreted data and contributed to the writing of the manuscript (including the figures). T. Madl conceived and performed NMR experiments, interpreted the NMR data and contributed to the writing of the manuscript (including the NMR figures). L.C. conducted initial NMR experiments. P.N. performed crystallographic data collection, data processing and structure refinement, and contributed to the interpretation of the crystallographic data. T. Monecke carried out crystallographic data collection. R.F. conducted molecular replacement for the binary CRM1–RanGTP complex. M.S. conceived NMR experiments, interpreted NMR data and contributed to the writing of the manuscript. D.G. conceived biochemical and crystallization experiments, prepared home-made affinity matrices, interpreted data and wrote the manuscript. All authors commented on the paper.

Corresponding author

Correspondence to Dirk Görlich.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results (Supplementary Figures 1–7 and Supplementary Table 1) and Supplementary Methods (PDF 5512 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Güttler, T., Madl, T., Neumann, P. et al. NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat Struct Mol Biol 17, 1367–1376 (2010). https://doi.org/10.1038/nsmb.1931

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1931

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing