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:

Tertiary interactions within the ribosomal exit tunnel

Nature Structural & Molecular Biology volume 16, pages 405–411 (2009)Cite this article

Abstract

Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a β-hairpin and an α-helical hairpin from the cytosolic N terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.

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: The T1 domain and experimental design.
Figure 2: Cross-linking and accessibility assays for β- and α-hairpins.
Figure 3: Accessibility-dependent probability of cross-linking.
Figure 4: T1 domain mutants.

Similar content being viewed by others

References

  1. Kosolapov, A., Tu, L., Wang, J. & Deutsch, C. Structure acquisition of the T1 domain of Kv1.3 during biogenesis. Neuron 44, 295–307 (2004).

    Article  CAS  Google Scholar 

  2. Lu, J. & Deutsch, C. Secondary structure formation of a transmembrane segment in Kv channels. Biochemistry 44, 8230–8243 (2005).

    Article  CAS  Google Scholar 

  3. Tu, L., Wang, J. & Deutsch, C. Biogenesis of the T1–S1 linker of voltage-gated K+ channels. Biochemistry 46, 8075–8084 (2007).

    Article  CAS  Google Scholar 

  4. Woolhead, C.A., McCormick, P.J. & Johnson, A.E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725–736 (2004).

    Article  CAS  Google Scholar 

  5. Mingarro, I., Nilsson, I., Whitley, P. & von Heijne, G. Different conformations of nascent polypeptides during translocation across the ER membrane. BMC Cell Biol. 1, 3 (2000).

    Article  CAS  Google Scholar 

  6. Kowarik, M., Kung, S., Martoglio, B. & Helenius, A. Protein folding during cotranslational translocation in the endoplasmic reticulum. Mol. Cell 10, 769–778 (2002).

    Article  CAS  Google Scholar 

  7. Hardesty, B. & Kramer, G. Folding of a nascent peptide on the ribosome. Prog. Nucleic Acid Res. Mol. Biol. 66, 41–66 (2001).

    Article  CAS  Google Scholar 

  8. Matlack, K.E. & Walter, P. The 70 carboxyl-terminal amino acids of nascent secretory proteins are protected from proteolysis by the ribosome and the protein translocation apparatus of the endoplasmic reticulum membrane. J. Biol. Chem. 270, 6170–6180 (1995).

    Article  CAS  Google Scholar 

  9. Ban, N., Nissen, P., Hansen, J., Moore, P.B. & Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920 (2000).

    Article  CAS  Google Scholar 

  10. Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

    Article  CAS  Google Scholar 

  11. Menetret, J.F. et al. The structure of ribosome-channel complexes engaged in protein translocation. Mol. Cell 6, 1219–1232 (2000).

    Article  CAS  Google Scholar 

  12. Beckmann, R. et al. Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361–372 (2001).

    Article  CAS  Google Scholar 

  13. Minor, D.L. et al. The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel. Cell 102, 657–670 (2000).

    Article  CAS  Google Scholar 

  14. Kreusch, A., Pfaffinger, P.J., Stevens, C.F. & Choe, S. Crystal structure of the tetramerization domain of the Shaker potassium channel. Nature 392, 945–948 (1998).

    Article  CAS  Google Scholar 

  15. Li, M., Jan, Y.N. & Jan, L.Y. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channels. Science 257, 1225–1230 (1992).

    Article  CAS  Google Scholar 

  16. Shen, N.V., Chen, X., Boyer, M.M. & Pfaffinger, P. Deletion analysis of K+ channel assembly. Neuron 11, 67–76 (1993).

    Article  CAS  Google Scholar 

  17. Xu, J., Yu, W., Jan, J.N., Jan, L. & Li, M. Assembly of voltage-gated potassium channels. Conserved hydrophilic motifs determine subfamily-specific interactions between the α-subunits. J. Biol. Chem. 270, 24761–24768 (1995).

    Article  CAS  Google Scholar 

  18. Gu, C., Jan, Y.N. & Jan, L.Y. A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels. Science 301, 646–649 (2003).

    Article  CAS  Google Scholar 

  19. Cushman, S.J. et al. Voltage dependent activation of potassium channels is coupled to T1 domain structure. Nat. Struct. Biol. 7, 403–407 (2000).

    Article  CAS  Google Scholar 

  20. Kurata, H.T. et al. Amino-terminal determinants of U-type inactivation of voltage-gated K+ channels. J. Biol. Chem. 277, 29045–29053 (2002).

    Article  CAS  Google Scholar 

  21. Wang, G. & Covarrubias, M. Voltage-dependent gating rearrangements in the intracellular T1–T1 interface of a K+ channel. J. Gen. Physiol. 127, 391–400 (2006).

    Article  CAS  Google Scholar 

  22. Lu, J., Robinson, J.M., Edwards, D. & Deutsch, C. T1–T1 interactions occur in ER membranes while nascent Kv peptides are still attached to ribosomes. Biochemistry 40, 10934–10946 (2001).

    Article  CAS  Google Scholar 

  23. Kosolapov, A. & Deutsch, C. Folding of the voltage-gated K+ channel T1 recognition domain. J. Biol. Chem. 278, 4305–4313 (2003).

    Article  CAS  Google Scholar 

  24. Lu, J. & Deutsch, C. Pegylation: a method for assessing topological accessibilities in Kv1.3. Biochemistry 40, 13288–13301 (2001).

    Article  CAS  Google Scholar 

  25. Robinson, J.M. & Deutsch, C. Coupled tertiary folding and oligomerization of the T1 domain of Kv channels. Neuron 45, 223–232 (2005).

    Article  CAS  Google Scholar 

  26. Kolb, V.A., Makeyev, E.V. & Spirin, A.S. Folding of firefly luciferase during translation in a cell-free system. EMBO J. 13, 3631–3637 (1994).

    Article  CAS  Google Scholar 

  27. Makeyev, E.V., Kolb, V.A. & Spirin, A.S. Enzymatic activity of the ribosome-bound nascent polypeptide. FEBS Lett. 378, 166–170 (1996).

    Article  CAS  Google Scholar 

  28. Voss, N.R., Gerstein, M., Steitz, T.A. & Moore, P.B. The geometry of the ribosomal polypeptide exit tunnel. J. Mol. Biol. 360, 893–906 (2006).

    Article  CAS  Google Scholar 

  29. Lu, J. & Deutsch, C. Folding zones inside the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 12, 1123–1129 (2005).

    Article  CAS  Google Scholar 

  30. Lu, J., Kobertz, W.R. & Deutsch, C. Mapping the electrostatic potential within the ribosomal exit tunnel. J. Mol. Biol. 371, 1378–1391 (2007).

    Article  CAS  Google Scholar 

  31. Steitz, T.A. A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Biol. 9, 242–253 (2008).

    Article  CAS  Google Scholar 

  32. Gabashvili, I.S. et al. The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Mol. Cell 8, 181–188 (2001).

    Article  CAS  Google Scholar 

  33. Nakatogawa, H. & Ito, K. The ribosomal exit tunnel functions as a discriminating gate. Cell 108, 629–636 (2002).

    Article  CAS  Google Scholar 

  34. Berisio, R. et al. Structural insight into the role of the ribosomal tunnel in cellular regulation. Nat. Struct. Biol. 10, 366–370 (2003).

    Article  CAS  Google Scholar 

  35. Tu, D., Blaha, G., Moore, P.B. & Steitz, T.A. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell 121, 257–270 (2005).

    Article  CAS  Google Scholar 

  36. Daggett, V. & Fersht, A.R. Is there a unifying mechanism for protein folding? Trends Biochem. Sci. 28, 18–25 (2003).

    Article  CAS  Google Scholar 

  37. Chandler, D. Interfaces and the driving force of hydrophobic assembly. Nature 437, 640–647 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Horn, J. Frank and U. Hartl for careful reading of the manuscript and R. Horn for helpful discussion. This research was funded by the US National Institutes of Health grant GM 52302 to C.D.

Author information

Authors and Affiliations

  1. Department of Physiology, University of Pennsylvania, Philadelphia, 19104-6085, Pennsylvania, USA

    Andrey Kosolapov & Carol Deutsch

Authors
  1. Andrey Kosolapov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carol Deutsch
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.K. performed the experiments; A.K. and C.D. designed the research, interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Carol Deutsch.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Results (PDF 333 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kosolapov, A., Deutsch, C. Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol Biol 16, 405–411 (2009). https://doi.org/10.1038/nsmb.1571

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1571

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