Article | Published:

Structure of the 40S–ABCE1 post-splitting complex in ribosome recycling and translation initiation

Nature Structural & Molecular Biology volume 24, pages 453460 (2017) | Download Citation

Abstract

The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S–ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. 1.

    & Structure and mechanism of ABC transporters. Curr. Opin. Struct. Biol. 12, 754–760 (2002).

  2. 2.

    & Evolution of the ATP-binding cassette (ABC) transporter superfamily in vertebrates. Annu. Rev. Genomics Hum. Genet. 6, 123–142 (2005).

  3. 3.

    , & ABC transporters: the power to change. Nat. Rev. Mol. Cell Biol. 10, 218–227 (2009).

  4. 4.

    Invited review: architectures and mechanisms of ATP binding cassette proteins. Biopolymers 105, 492–504 (2016).

  5. 5.

    , & Termination and post-termination events in eukaryotic translation. Adv. Protein Chem. Struct. Biol. 86, 45–93 (2012).

  6. 6.

    , & Structural view on recycling of archaeal and eukaryotic ribosomes after canonical termination and ribosome rescue. Curr. Opin. Struct. Biol. 22, 786–796 (2012).

  7. 7.

    & Tying up loose ends: ribosome recycling in eukaryotes and archaea. Trends Biochem. Sci. 38, 64–74 (2013).

  8. 8.

    & Translation drives mRNA quality control. Nat. Struct. Mol. Biol. 19, 594–601 (2012).

  9. 9.

    et al. Structural organization of essential iron-sulfur clusters in the evolutionarily highly conserved ATP-binding cassette protein ABCE1. J. Biol. Chem. 282, 14598–14607 (2007).

  10. 10.

    , & X-ray structure of the complete ABC enzyme ABCE1 from Pyrococcus abyssi. J. Biol. Chem. 283, 7962–7971 (2008).

  11. 11.

    et al. Structural basis of highly conserved ribosome recycling in eukaryotes and archaea. Nature 482, 501–506 (2012).

  12. 12.

    et al. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1. Cell Rep. 8, 59–65 (2014).

  13. 13.

    , , , & Structural basis for stop codon recognition in eukaryotes. Nature 524, 493–496 (2015).

  14. 14.

    et al. The role of ABCE1 in eukaryotic posttermination ribosomal recycling. Mol. Cell 37, 196–210 (2010).

  15. 15.

    & Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast. Proc. Natl. Acad. Sci. USA 108, E1392–E1398 (2011).

  16. 16.

    , , , & Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 1804–1817 (2011).

  17. 17.

    , , & Dom34-Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress. EMBO J. 33, 265–276 (2014).

  18. 18.

    et al. Ribosome recycling depends on a mechanistic link between the FeS cluster domain and a conformational switch of the twin-ATPase ABCE1. Proc. Natl. Acad. Sci. USA 108, 3228–3233 (2011).

  19. 19.

    & Dom34 rescues ribosomes in 3′ untranslated regions. Cell 156, 950–962 (2014).

  20. 20.

    , , , & Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3′UTRs in vivo. Cell 162, 872–884 (2015).

  21. 21.

    et al. The essential ATP-binding cassette protein RLI1 functions in translation by promoting preinitiation complex assembly. J. Biol. Chem. 279, 42157–42168 (2004).

  22. 22.

    & The essential Drosophila ATP-binding cassette domain protein, pixie, binds the 40 S ribosome in an ATP-dependent manner and is required for translation initiation. J. Biol. Chem. 282, 14752–14760 (2007).

  23. 23.

    , , & Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol. Cell 51, 249–264 (2013).

  24. 24.

    eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem. Sci. 31, 553–562 (2006).

  25. 25.

    et al. Molecular architecture of a eukaryotic translational initiation complex. Science 342, 1240585 (2013).

  26. 26.

    et al. Structural snapshots of actively translating human ribosomes. Cell 161, 845–857 (2015).

  27. 27.

    et al. Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26, 2421–2431 (2007).

  28. 28.

    et al. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J. 23, 1008–1019 (2004).

  29. 29.

    et al. Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome. Nat. Struct. Mol. Biol. 18, 715–720 (2011).

  30. 30.

    et al. Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry. Nat. Commun. 7, 13248 (2016).

  31. 31.

    & Snapshots of the maltose transporter during ATP hydrolysis. Proc. Natl. Acad. Sci. USA 108, 15152–15156 (2011).

  32. 32.

    , , & Structure of AMP-PNP-bound BtuCD and mechanism of ATP-powered vitamin B12 transport by BtuCD-F. Nat. Struct. Mol. Biol. 21, 1097–1099 (2014).

  33. 33.

    et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl. Acad. Sci. USA 106, 1063–1068 (2009).

  34. 34.

    , , & Crystal structures of ribosome anti-association factor IF6. Nat. Struct. Biol. 7, 1156–1164 (2000).

  35. 35.

    et al. Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3′ end of aberrant mRNA. Mol. Cell 46, 518–529 (2012).

  36. 36.

    Function and biogenesis of iron-sulphur proteins. Nature 460, 831–838 (2009).

  37. 37.

    et al. Growth and cell survival are unevenly impaired in pixie mutant wing discs. Development 132, 5411–5424 (2005).

  38. 38.

    et al. Sculpting the proteome with AAA(+) proteases and disassembly machines. Cell 119, 9–18 (2004).

  39. 39.

    & Meiotic clade AAA ATPases: protein polymer disassembly machines. J. Mol. Biol. 428, 1897–1911 (2016).

  40. 40.

    et al. Conformational differences between open and closed states of the eukaryotic translation initiation complex. Mol. Cell 59, 399–412 (2015).

  41. 41.

    & Analysis of eukaryotic translation in purified and semipurified systems. Curr. Protoc. Cell. Biol. 8, 11.19.1–11.19.26 (2001).

  42. 42.

    et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

  43. 43.

    , , & Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

  44. 44.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  45. 45.

    et al. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science 334, 1524–1529 (2011).

  46. 46.

    et al. ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. Mol. Cell 10, 139–149 (2002).

  47. 47.

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

  48. 48.

    et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015).

  49. 49.

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

  50. 50.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  51. 51.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

  52. 52.

    et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

  53. 53.

    , , , & Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome. Cell 157, 823–831 (2014).

  54. 54.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  55. 55.

    et al. Structure of a yeast 40S–Eif1–Eif1A–Eif3–Eif3j initiation complex. Nat. Struct. Mol. Biol. 22, 269–271 (2015).

Download references

Acknowledgements

The authors thank K. Kiosze-Becker, E. Nürenberg-Goloub, B. Hetzert, C. Le Gal and C. Thomas for helpful suggestions on the manuscript and C. Ungewickell, S. Lange and S. Lamberth for technical assistance. This work was supported by the German Research Council (grants SFB 902 to R.T., SFB 646 to R.B. and T.B., FOR 1805 to R.B., GRK 1721 to R.B.). R.B. acknowledges support by the Center for Integrated Protein Science Munich (CiPS-M) and the European Research Council (Advanced Grants CRYOTRANSLATION). The Cluster of Excellence–Macromolecular Complexes (EXC 115 to R.T.) supported the work. M.G. and C.S. were supported by Boehringer Ingelheim Fonds PhD fellowships. We thank the Leibniz-Rechenzentrum Munich (LRZ) for providing computational services and support.

Author information

Author notes

    • André Heuer
    •  & Milan Gerovac

    These authors contributed equally to this work.

Affiliations

  1. Gene Center and Center of Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany.

    • André Heuer
    • , Christian Schmidt
    • , Anne Preis
    • , Otto Berninghausen
    • , Thomas Becker
    •  & Roland Beckmann
  2. Institute of Biochemistry, Biocenter, Goethe University, Frankfurt/Main, Germany.

    • Milan Gerovac
    • , Simon Trowitzsch
    •  & Robert Tampé
  3. Institute for Molecular Genetics and Cellular Microbiology, Biocenter, Goethe University, Frankfurt/Main, Germany.

    • Peter Kötter

Authors

  1. Search for André Heuer in:

  2. Search for Milan Gerovac in:

  3. Search for Christian Schmidt in:

  4. Search for Simon Trowitzsch in:

  5. Search for Anne Preis in:

  6. Search for Peter Kötter in:

  7. Search for Otto Berninghausen in:

  8. Search for Thomas Becker in:

  9. Search for Roland Beckmann in:

  10. Search for Robert Tampé in:

Contributions

M.G., A.H., T.B., R.B. and R.T. designed the study. M.G. developed the preparation of the post-splitting complex and performed all functional assays. M.G. and P.K. conducted the plasmid shuffling experiment. M.G. and S.T. designed the NTPase assays. M.G. and A.H. prepared the EM samples. A.P. and A.H. prepared the initiation complex. A.H. and O.B. collected and A.H. processed the cryo-EM data. C.S., A.H. and T.B. built and refined the model. C.S., T.B., A.H. and M.G. analyzed and interpreted the structures. M.G., T.B., A.H., S.T., R.B. and R.T. wrote the manuscript. R.T. initiated and R.B. and R.T. conceived the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Roland Beckmann or Robert Tampé.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Excel files

  1. 1.

    Supplementary Data Set 1

Videos

  1. 1.

    Transition of ABCE1 from the open to the closed state.

    In the free state, ABCE1 is in the open state with two ADPs occluded. After binding to 80S pre-termination complexes (with eRF1 or Dom34 or Pelota), ABCE1 changes its conformation to an intermediate, semi-closed (pre-splitting) state. Here the nucleotide occupation is less clear. High and medium resolved cryo-EM structures showed density for a bound nucleotide in NBS I, whereas NBS II was rather empty. Occlusion of both NBSs with ATP leads to closure of the NBDs which is required for ribosome splitting. This closure is accompanied by rearrangement of the FeS cluster domain. In the post-splitting state, ABCE1 can be trapped on the 40S subunit in a closed conformation occluding two ATP (here AMP-PNP). The FeS cluster domain rotates into a binding cleft formed by h5, h44 and uS12. This conformation is mainly stabilized by interactions of Pro30 to uS12 and Arg7 to h5. Moreover, the cantilever helix unwinds and Tyr301 contacts the backbone of Asn78. The HLH motif interacts with the junction of h8 and h14 mainly via Ser150. Additional conformational changes occur in the C-terminal helix of eS24, which contacts NBD1 (Gln262) in post-splitting state. Only minor conformational changes occur for the hinge 2 motif, which contacts the h5 and h15 junction via Arg573 and Ser588. A zoom into NBS I shows how the nucleotide is occluded by ABC motifs. Open (homology model based on PDB: 3BK7), semi-closed (PDB: 3J16), and closed state (this study) of ABCE1 were morphed using UCSF-Chimera.

  2. 2.

    Mechanism of ABCE1-dependent ribosome splitting.

    In the pre-splitting state, ABCE1 is bound to an 80S ribosome occupied with an A-site factor (here eRF1). The FeS cluster domain interacts with the C-terminal domain of eRF1 (or Pelota). In phase 1, ATP-binding leads to closing of the NBDs and rearrangement of the FeS cluster domain in direction of the bound A-site factor. It acts as a wedge, which leads to destabilization of the 80S ribosome. In phase 2, after dissociation of the 60S, the FeS cluster domain locks into its final position and prevents formation of an intersubunit bridge B5 involving uL14 (green), thus acting as an anti-association factor. Isolated densities of the 60S subunit (dark grey), eRF1 (blue), pre-splitting state of ABCE1 (yellow) as well as respective models were taken from EMD-2598 (PDB: 4CRM).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3396

Further reading