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:

Structure of the nuclear transport complex karyopherin-β2–Ran˙GppNHp

Nature volume 399, pages 230–237 (1999)Cite this article

Abstract

Transport factors in the karyopherin-β (also called importin-β) family mediate the movement of macromolecules in nuclear–cytoplasmic transport pathways. Karyopherin-β2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran˙GTP binds karyopherin-β2 and dissociates the substrate. Here we present the 3.0 Å structure of the karyopherin-β2–Ran˙GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-β2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino-terminal arch and substrate-binding activity is mapped to the carboxy-terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran˙GppNHp in the complex shows extensive structural rearrangement, compared to Ran˙GDP, in regions contacting karyopherin-β2. This provides a structural basis for the specificity of the karyopherin-β family for the GTP-bound state of Ran, as well as a rationale for interactions of the karyopherin–Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.

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: Stereoview of a ribbon drawing of Kap-β2 bound to Ran˙GppNHp.
Figure 2: Kap-β2 contains eighteen HEAT repeats.
Figure 3: Structural differences between Ran˙GppNHp and Ran˙GDP.
Figure 4: Kap-β2–Ran˙GppNHp interface.
Figure 5: Kap-β2–Ran and RBD1–Ran interfaces are mostly non-overlapping but adjacent in the Ran structure.
Figure 6: Electrostatic surface potential and sequence conservation mapped ontothe molecular surface of Kap-β2 in the Kap-β2–Ran˙GppNHp complex.

Similar content being viewed by others

References

  1. Pemberton, L. F., Blobel, G. & Rosenblum, J. S. Transport routes through the nuclear pore complex. Curr. Opin. Cell Biol. 10, 392– 399 (1998).

    Article  CAS  Google Scholar 

  2. Nehrbass, U. & Blobel, G. Role of the nuclear transport factor p10 in nuclear import. Science 272, 120– 122 (1996).

    Article  ADS  CAS  Google Scholar 

  3. Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M. & Gorlich, D. NTF2 mediates nuclear import of Ran. EMBO J. 17, 6587–6598 (1998).

    Article  CAS  Google Scholar 

  4. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell 94, 193–204 ( 1998).

    Article  CAS  Google Scholar 

  5. Albertini, M., Pemberton, L. F., Rosenblum, J. S. & Blobel, G. Anovel nuclear import pathway for the transcription factor TFIIS. J. Cell Biol. 143, 1447–1455 (1998).

    Article  CAS  Google Scholar 

  6. Kaffman, A., Rank, N. M. & O'Shea, E. K. Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes Dev. 12, 2673–2683 (1998).

    Article  CAS  Google Scholar 

  7. Kaffman, A., Rank, N. M., O'Neill, E. M., Huang, L. S. & O'Shea, E. K. The receptor MsN5 exports the phosphorylated transcription factor Pho4 out of the nucleus. Nature 396, 482–486 ( 1998).

    Article  ADS  CAS  Google Scholar 

  8. Rexach, M. & Blobel, G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683– 692 (1995).

    Article  CAS  Google Scholar 

  9. Siomi, M. C. et al. Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J.Cell Biol. 138, 1181– 1192 (1997).

    Article  CAS  Google Scholar 

  10. Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W. & Gorlich, 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 

  11. Bischoff, F. R. & Ponstingl, H. Catalysis of guanine nucleotide exchange of Ran by RCC1 and stimulation of hydrolysis of Ran-bound GTP by Ran-GAP1. Methods Enzymol. 257, 135–144 (1995).

    Article  CAS  Google Scholar 

  12. Bonifaci, N., Moroianu, J., Radu, A. & Blobel, G. Karyopherin beta2 mediates nuclear import of a mRNA binding protein. Proc. Natl Acad. Sci. USA 94, 5055–5060 ( 1997).

    Article  ADS  CAS  Google Scholar 

  13. Pollard, V. W. et al. Anovel receptor-mediated nuclear protein import pathway. Cell 86, 985–994 ( 1996).

    Article  CAS  Google Scholar 

  14. Fridell, R. A., Truant, R., Thorne, L., Benson, R. E. & Cullen, B. R. Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J. Cell Sci. 110, 1325–1331 (1997).

    CAS  PubMed  Google Scholar 

  15. Andrade, M. A. & Bork, P. HEAT repeats in Huntington's disease protein. Nature Genet. 11, 115– 116 (1995).

    Article  CAS  Google Scholar 

  16. Stewart, M., Kent, H. M. & McCoy, A. J. The structure of the Q69L mutant of GDP-Ran shows a major conformational change in the switchII loop that accounts for its failure to bind nuclear transport factor2 (NTF2). J. Mol. Biol. 284, 1517–1527 (1998).

    Article  CAS  Google Scholar 

  17. Scheffzek, K., Klebe, C., Fritz-Wolf, K., Kabsch, W. & Wittinghofer, A. Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form. Nature 374, 378–381 (1995).

    Article  ADS  CAS  Google Scholar 

  18. Sheldrick, G. M., Dauter, Z., Wilson, K. S., Hope, H. & Sieker, L. C. The application of direct methods and Patterson interpretation to high-resolution native protein data. Acta Crystallogr. D 49, 18–23 (1993).

    Article  CAS  Google Scholar 

  19. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A. & Barford, D. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 randomly repeated HEAT motifs. Cell 96, 99–110 ( 1999).

    Article  CAS  Google Scholar 

  20. Huber, A. H., Nelson, W. J. & Weis, W. I. Three-dimensional structure of the armadillo repeat region of β-catenin. Cell 90, 871– 882 (1997).

    Article  CAS  Google Scholar 

  21. Malik, H. S., Eickbush, T. H. & Goldfarb, D. S. Evolutionary specialization of the nuclear targeting apparatus. Proc. Natl Acad. Sci. USA 94, 13738–3742 (1997).

    Article  ADS  CAS  Google Scholar 

  22. Richards, S. A., Lounsbury, K. M. & Macara, I. G. The C terminus of the nuclear RAN/TC4 GTPase stabilizes the GDP-bound state and mediates interactions with RCC1, RAN-GAP, and HTF9A/RANBP1. J. Biol. Chem. 270, 14405– 14411 (1995).

    Article  CAS  Google Scholar 

  23. Vetter, I. R., Nowak, C., Nishimoto, T., Kuhlmann, J. & Wittinghofer, A. Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature 398, 39–46 ( 1999).

    Article  ADS  CAS  Google Scholar 

  24. Pai, E. F. et al. Structure of the guanine-nucleotide-binding domain of the Ha- ras oncogene product p21 in the triphosphate conformation. Nature 341, 209–214 ( 1989).

    Article  ADS  CAS  Google Scholar 

  25. Hidea, M. et al. Amonoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells. J. Cell Biol. 144, 645– 655 (1999).

    Article  Google Scholar 

  26. Kutay, U., Izaurralde, E., Bischoff, F. R., Mattaj, I. W. & Gorlich, D. Dominant-negative mutants of important-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16, 1153–1163 (1997).

    Article  CAS  Google Scholar 

  27. Kose, S., Imamoto, N., Tachibana, T., Shimamoto, T. & Yoneda, Y. Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex. J. Cell Biol. 139, 841–849 ( 1997).

    Article  CAS  Google Scholar 

  28. Chi, N. C., Adam, E. J. H. & Adam, S. A. Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97. J. Biol. Chem. 272, 6818–6822 (1997).

    Article  CAS  Google Scholar 

  29. Moroianu, J., Blobel, G. & Radu, A. Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta. Proc. Natl Acad. Sci. USA 93, 7059 –7062 (1996).

    Article  ADS  CAS  Google Scholar 

  30. Enenkel, C., Schulke, N. & Blobel, G. Expression in yeast of binding regions of karyopherins alpha and beta inhibits nuclear import and cell growth. Proc. Natl Acad. Sci. USA 93, 12986–12991 (1996).

    Article  ADS  CAS  Google Scholar 

  31. Gorlich, D., Pante, N., Kutay, U., Aebi, U. & Bischoff, F. R. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584–5594 (1996).

    Article  CAS  Google Scholar 

  32. Floer, M. & Blobel, G. The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein. J. Biol. Chem. 271, 5313– 5316 (1996).

    Article  CAS  Google Scholar 

  33. Lounsbury, K. M. & Macara, I. G. Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by karyopherin beta. J. Biol. Chem. 272, 551– 555 (1997).

    Article  CAS  Google Scholar 

  34. Scheffzek, K. et al. The Ras-RasGAP complex: Structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338 (1997).

    Article  CAS  Google Scholar 

  35. Rittinger, K., Walker, P. A., Eccleston, J. F., Smerdon, S. J. & Gamblin, S. J. Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389, 758– 762 (1997).

    Article  ADS  CAS  Google Scholar 

  36. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D. & Kuriyan, K. The structural basis of the activation of Ras by Sos. Nature 394, 337–343 (1998).

    Article  ADS  CAS  Google Scholar 

  37. Coutavas, E., Ren, M., Oppenheim, J. D., E'Eustachio, P. & Rush, M. G. Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4. Nature 366, 585–587 ( 1993).

    Article  ADS  CAS  Google Scholar 

  38. Wu, J., Matunis, M. J., Kraemer, D., Blobel, G. & Coutavas, E. Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J. Biol. Chem. 270, 14209–14213 ( 1995).

    Article  CAS  Google Scholar 

  39. Yokoyama, N. et al. Agiant nucleopore protein that binds Ran/TC4. Nature 376, 184–188 ( 1995).

    Article  ADS  CAS  Google Scholar 

  40. Bischoff, F. R. & Gorlich, D. RanBP1 is crucial for the release of RanGTP from importin beta-related nuclear transport factors. FEBS Lett. 419, 249–254 (1997).

    Article  CAS  Google Scholar 

  41. Kuhlmann, J., Macara, I. & Wittinghofer, A. Dynamic and equilibrium studies on the interaction of Ran with its effector, RanBP1. Biochemistry 36, 12027–12035 (1997).

    Article  CAS  Google Scholar 

  42. Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L. & Clardy, J. Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. Science 251, 839–842 ( 1991).

    Article  ADS  Google Scholar 

  43. Westbrook, E. M. & Naday, I. Charge-coupled device-based area detectors. Methods Enzymol. 276, 244 –268 (1997).

    Article  CAS  Google Scholar 

  44. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation model. Methods Enzymol. 276, 307–326 ( 1997).

    Article  CAS  Google Scholar 

  45. CCP4. The CCP4 suite: programs for X-ray crystallography. Acta Crystallogr. D 50, 760– 776 (1994).

  46. Jones, T. A., Cowan, S. W. & Kjelgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 ( 1991).

    Article  Google Scholar 

  47. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  48. Kraulis, P. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946 –950 (1991).

    Article  Google Scholar 

  49. Merritt, E. A. & Murphy, M. E. P. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallog. D 50, 869–873 (1994).

    Article  CAS  Google Scholar 

  50. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Jeffreys and N. Pavletich of MSKCC and J. Kuriyan of RU for the use of X-ray data collection facilities; R. Sweet and L. Berman of NSLS and M. Rosen for help with synchrotron data collection; J. Kuriyan and T. Sakmar for use of computation facility; N. Bonifaci for the clone of Kap β2; and M.Rosen, E. Conti, J. Bonanno, P. Jeffreys, P. Adams, K. Reinisch, J. Helmers, L. Pemberton, J. Rosenblum, M. Floer, M. Rout and J. Goldberg for advice and help. Y.M.C. is supported by a Life Sciences Research Foundation Fellowship sponsored by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, 10021, New York, USA

    Yuh Min Chook & Günter Blobel

Authors
  1. Yuh Min Chook
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Günter Blobel
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chook, Y., Blobel, G. Structure of the nuclear transport complex karyopherin-β2–Ran˙GppNHp . Nature 399, 230–237 (1999). https://doi.org/10.1038/20375

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/20375

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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