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Cotranslational folding of spectrin domains via partially structured states

Nature Structural & Molecular Biology volume 24, pages 221–225 (2017)Cite this article

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Abstract

How do the key features of protein folding, elucidated from studies on native, isolated proteins, manifest in cotranslational folding on the ribosome? Using a well-characterized family of homologous α-helical proteins with a range of biophysical properties, we show that spectrin domains can fold vectorially on the ribosome and may do so via a pathway different from that of the isolated domain. We use cryo-EM to reveal a folded or partially folded structure, formed in the vestibule of the ribosome. Our results reveal that it is not possible to predict which domains will fold within the ribosome on the basis of the folding behavior of isolated domains; instead, we propose that a complex balance of the rate of folding, the rate of translation and the lifetime of folded or partly folded states will determine whether folding occurs cotranslationally on actively translating ribosomes.

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Figure 1: Three-helix bundle structure of spectrin domains.
Figure 2: Investigating cotranslational folding using the AP assay.
Figure 3: Force profiles.
Figure 4: Visualization of the R16 spectrin domain at the ribosomal tunnel exit.
Figure 5: Rigid body fit of the NMR structure of the R16 domain to the cryo-EM density map showing equivalent locations of R15 key folding residues.

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References

  1. Braselmann, E., Chaney, J.L. & Clark, P.L. Folding the proteome. Trends Biochem. Sci. 38, 337–344 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Joh, N.H. et al. De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 346, 1520–1524 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Song, W.J. & Tezcan, F.A. A designed supramolecular protein assembly with in vivo enzymatic activity. Science 346, 1525–1528 (2014).

    CAS  PubMed  Google Scholar 

  4. Koga, N. et al. Principles for designing ideal protein structures. Nature 491, 222–227 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Hingorani, K.S. & Gierasch, L.M. Comparing protein folding in vitro and in vivo: foldability meets the fitness challenge. Curr. Opin. Struct. Biol. 24, 81–90 (2014).

    CAS  PubMed  Google Scholar 

  6. Frydman, J., Erdjument-Bromage, H., Tempst, P. & Hartl, F.U. Co-translational domain folding as the structural basis for the rapid de novo folding of firefly luciferase. Nat. Struct. Biol. 6, 697–705 (1999).

    CAS  PubMed  Google Scholar 

  7. Kim, S.J. et al. Protein folding. Translational tuning optimizes nascent protein folding in cells. Science 348, 444–448 (2015).

    CAS  PubMed  Google Scholar 

  8. Evans, M.S., Sander, I.M. & Clark, P.L. Cotranslational folding promotes β-helix formation and avoids aggregation in vivo. J. Mol. Biol. 383, 683–692 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 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).

    CAS  PubMed  Google Scholar 

  10. Bhushan, S. et al. SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center. PLoS Biol. 9, e1000581 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bhushan, S. et al. α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 17, 313–317 (2010).

    CAS  PubMed  Google Scholar 

  12. Nilsson, O.B. et al. Cotranslational protein folding inside the ribosome exit tunnel. Cell Reports 12, 1533–1540 (2015).

    CAS  PubMed  Google Scholar 

  13. O'Brien, E.P., Hsu, S.T., Christodoulou, J., Vendruscolo, M. & Dobson, C.M. Transient tertiary structure formation within the ribosome exit port. J. Am. Chem. Soc. 132, 16928–16937 (2010).

    CAS  PubMed  Google Scholar 

  14. Tu, L., Khanna, P. & Deutsch, C. Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome. J. Mol. Biol. 426, 185–198 (2014).

    CAS  PubMed  Google Scholar 

  15. Kosolapov, A. & Deutsch, C. Tertiary interactions within the ribosomal exit tunnel. Nat. Struct. Mol. Biol. 16, 405–411 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Holtkamp, W. et al. Cotranslational protein folding on the ribosome monitored in real time. Science 350, 1104–1107 (2015).

    CAS  PubMed  Google Scholar 

  17. O'Brien, E.P., Christodoulou, J., Vendruscolo, M. & Dobson, C.M. New scenarios of protein folding can occur on the ribosome. J. Am. Chem. Soc. 133, 513–526 (2011).

    CAS  PubMed  Google Scholar 

  18. Ismail, N., Hedman, R., Lindén, M. & von Heijne, G. Charge-driven dynamics of nascent-chain movement through the SecYEG translocon. Nat. Struct. Mol. Biol. 22, 145–149 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Cymer, F. & von Heijne, G. Cotranslational folding of membrane proteins probed by arrest-peptide-mediated force measurements. Proc. Natl. Acad. Sci. USA 110, 14640–14645 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Ismail, N., Hedman, R., Schiller, N. & von Heijne, G. A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration. Nat. Struct. Mol. Biol. 19, 1018–1022 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Goldman, D.H. et al. Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo. Science 348, 457–460 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Nilsson, O.B., Müller-Lucks, A., Kramer, G., Bukau, B. & von Heijne, G. Trigger factor reduces the force exerted on the nascent chain by a cotranslationally folding protein. J. Mol. Biol. 428, 1356–1364 (2016).

    CAS  PubMed  Google Scholar 

  23. Nakatogawa, H. & Ito, K. Secretion monitor, SecM, undergoes self-translation arrest in the cytosol. Mol. Cell 7, 185–192 (2001).

    CAS  PubMed  Google Scholar 

  24. Yap, M.N. & Bernstein, H.D. The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. Mol. Cell 34, 201–211 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Tsai, A., Kornberg, G., Johansson, M., Chen, J. & Puglisi, J.D. The dynamics of SecM-induced translational stalling. Cell Reports 7, 1521–1533 (2014).

    CAS  PubMed  Google Scholar 

  26. Gumbart, J., Schreiner, E., Wilson, D.N., Beckmann, R. & Schulten, K. Mechanisms of SecM-mediated stalling in the ribosome. Biophys. J. 103, 331–341 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Butkus, M.E., Prundeanu, L.B. & Oliver, D.B. Translocon “pulling” of nascent SecM controls the duration of its translational pause and secretion-responsive secA regulation. J. Bacteriol. 185, 6719–6722 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Shimizu, Y., Kanamori, T. & Ueda, T. Protein synthesis by pure translation systems. Methods 36, 299–304 (2005).

    CAS  PubMed  Google Scholar 

  29. Batey, S. & Clarke, J. The folding pathway of a single domain in a multidomain protein is not affected by its neighbouring domain. J. Mol. Biol. 378, 297–301 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Batey, S. & Clarke, J. Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains. Proc. Natl. Acad. Sci. USA 103, 18113–18118 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Batey, S., Randles, L.G., Steward, A. & Clarke, J. Cooperative folding in a multi-domain protein. J. Mol. Biol. 349, 1045–1059 (2005).

    CAS  PubMed  Google Scholar 

  32. Wensley, B.G., Kwa, L.G., Shammas, S.L., Rogers, J.M. & Clarke, J. Protein folding: adding a nucleus to guide helix docking reduces landscape roughness. J. Mol. Biol. 423, 273–283 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Wensley, B.G. et al. Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family. Nature 463, 685–688 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Cabrita, L.D. et al. A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Nat. Struct. Mol. Biol. 23, 278–285 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Cabrita, L.D., Dobson, C.M. & Christodoulou, J. Protein folding on the ribosome. Curr. Opin. Struct. Biol. 20, 33–45 (2010).

    CAS  PubMed  Google Scholar 

  36. Kaiser, C.M., Goldman, D.H., Chodera, J.D., Tinoco, I. Jr. & Bustamante, C. The ribosome modulates nascent protein folding. Science 334, 1723–1727 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Scott, K.A., Randles, L.G. & Clarke, J. The folding of spectrin domains II: phi-value analysis of R16. J. Mol. Biol. 344, 207–221 (2004).

    CAS  PubMed  Google Scholar 

  38. Wensley, B.G., Gärtner, M., Choo, W.X., Batey, S. & Clarke, J. Different members of a simple three-helix bundle protein family have very different folding rate constants and fold by different mechanisms. J. Mol. Biol. 390, 1074–1085 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ingolia, N.T., Lareau, L.F. & Weissman, J.S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Young, R. & Bremer, H. Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate. Biochem. J. 160, 185–194 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Borgia, M.B. et al. Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins. Nature 474, 662–665 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Borgia, A. et al. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy. Nat. Commun. 3, 1195 (2012).

    PubMed  Google Scholar 

  43. Hill, S.A., Kwa, L.G., Shammas, S.L., Lee, J.C. & Clarke, J. Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered partners. J. Mol. Biol. 426, 21–35 (2014).

    CAS  PubMed  Google Scholar 

  44. Ke, S.H. & Madison, E.L. Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR method. Nucleic Acids Res. 25, 3371–3372 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Zheng, L., Baumann, U. & Reymond, J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004).

    PubMed  PubMed Central  Google Scholar 

  46. Bischoff, L., Berninghausen, O. & Beckmann, R. Molecular basis for the ribosome functioning as an L-tryptophan sensor. Cell Rep. 9, 469–475 (2014).

    CAS  PubMed  Google Scholar 

  47. Chen, J.Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007).

    CAS  PubMed  Google Scholar 

  48. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    CAS  PubMed  Google Scholar 

  49. Penczek, P.A., Frank, J. & Spahn, C.M. A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J. Struct. Biol. 154, 184–194 (2006).

    CAS  PubMed  Google Scholar 

  50. Scheres, S.H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Heuer for help preparing the figures. Supported by grants from the Swedish Cancer Foundation, the Swedish Research Council and the Knut and Alice Wallenberg Foundation (to G.v.H.); the Wellcome Trust (WT095195 to J.C.) and the European Research Council (ERC-2008-AdG 232648, to R.B.). J.C. is a Wellcome Trust Senior Research Fellow.

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Author notes

  1. Ola B Nilsson and Adrian A Nickson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics Stockholm University, Stockholm, Sweden

    Ola B Nilsson & Gunnar von Heijne

  2. Department of Chemistry, University of Cambridge, Cambridge, UK

    Adrian A Nickson, Jeffrey J Hollins, Annette Steward & Jane Clarke

  3. Gene Center and Center for Integrated Protein Science Munich (CiPS-M), University of Munich, Munich, Germany

    Stephan Wickles & Roland Beckmann

  4. Science for Life Laboratory, Stockholm University, Solna, Sweden

    Gunnar von Heijne

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Contributions

O.B.N. and A.A.N. designed and carried out the experiments; J.J.H. and A.S. characterized the purified proteins; S.W. and R.B. were responsible for the cryo-EM experiments; A.S. wrote the manuscript; G.v.H. and J.C. conceived and planned the investigation and wrote the manuscript.

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Correspondence to Gunnar von Heijne or Jane Clarke.

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Integrated supplementary information

Supplementary Figure 1 In vitro translation of the spectrin constructs.

(a) Schematic representation of the constructs used in the AP assays showing the Lep leader (blue), GSGS and SGSG linker sites (purple), the variable linker (green), the SecM stall site (grey) and the terminal Lep segment (orange). Constructs for β16, R16m5, R16m6, R16o15c and non-folding R15 and R16 all resemble R15. L, reported linker length (minimum L=21 amino acids, maximum L=61 amino acids). R15R16m5 and R153ProR16 resemble R15R16. Note that the R16 force profiles obtained with the LepB leader sequence are virtually identical (see Main text Fig 3 a,b). (b) R16 [L=27] and R16 [L=37] translated in the PURE system. Full-length (FL) and arrested (A) products are indicated. FLc, full-length control, where the crucial Pro at the C-terminal end of the SecM AP is mutated to Ala; Ac, arrest control, where the crucial Pro at the end of the AP is substituted with a stop codon.

Supplementary Figure 2 Reproducibility of the data.

Data for three separate repeats of wild-type R16 without the Lep leader are shown. Points in red are the full R16 trace (see Main text Fig 3b), points in green are repeats collected using an in vitro translation kit with a different lot number and points in blue are repeats collected in a different laboratory using an in vitro translation kit with a different lot number.

Supplementary Figure 3 Supplementary cryo-EM images.

(a) Resolution determination of the final reconstruction using Fourier shell correlation (FSC) indicating an average resolution of 4.8 Å. (b) Calculation of the local resolution using resmap (Kucukelbir, A. et al. Nat Methods 11, 63-65, 2014). (c) Superposition of the spectrin R16 domain (red) and the previously determined cryo-EM structure of the ADR1a zinc-finger domain (gold) in the ribosome exit tunnel12. Notably, the location in the exit tunnel of the C-terminus of the R16 domain is 25-30 Å away from the location of the C-terminus of ADR1a, consistent with the difference in linker lengths (L = 33 vs. 25 residues) used in the two constructs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 460 kb)

Rigid body fit of the NMR structure of the R16 domain to the cryo-EM density map.

Rigid body fit of the NMR structure of the R16 domain colored according to r.m.s. deviation (blue, 0.5–1.9; white, 2–3.9; red, ≥4.0 Å) to the cryo-EM density map (Fig. 4c). (MP4 5049 kb)

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Nilsson, O., Nickson, A., Hollins, J. et al. Cotranslational folding of spectrin domains via partially structured states. Nat Struct Mol Biol 24, 221–225 (2017). https://doi.org/10.1038/nsmb.3355

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