Mechanism of completion of peptidyltransferase centre assembly in eukaryotes

  1. Vasileios Kargas
  2. Pablo Castro-Hartmann
  3. Norberto Escudero-Urquijo
  4. Kyle Dent
  5. Christine Hilcenko
  6. Carolin Sailer
  7. Gertrude Zisser
  8. Maria J Marques-Carvalho
  9. Simone Pellegrino
  10. Leszek Wawiórka
  11. Stefan MV Freund
  12. Jane L Wagstaff
  13. Antonina Andreeva
  14. Alexandre Faille
  15. Edwin Chen
  16. Florian Stengel
  17. Helmut Bergler
  18. Alan John Warren  Is a corresponding author
  1. Cambridge Institute for Medical Research, United Kingdom
  2. University of Konstanz, Germany
  3. University of Graz, Austria
  4. MRC Laboratory of Molecular Biology, United Kingdom
  5. University of Leeds, United Kingdom

Abstract

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilizes helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

Data availability

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with accession numbers EMD-4558, EMD-4559, EMD-4560, EMD-4636, EMD-4884 and EMD-4630. Atomic coordinates have been deposited in the Protein Data Bank, with entry codes 6QIF, 6QIJ, 6QIK, 6QTZ, 6RI5 and 6QT0.

The following data sets were generated

Article and author information

Author details

  1. Vasileios Kargas

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Pablo Castro-Hartmann

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Norberto Escudero-Urquijo

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8201-5884
  4. Kyle Dent

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Christine Hilcenko

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Carolin Sailer

    Department of Biology, University of Konstanz, Konstanz, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Gertrude Zisser

    Institute of Molecular Bioscience, University of Graz, Graz, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Maria J Marques-Carvalho

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  9. Simone Pellegrino

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Leszek Wawiórka

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  11. Stefan MV Freund

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  12. Jane L Wagstaff

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  13. Antonina Andreeva

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Alexandre Faille

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Edwin Chen

    Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0742-9734
  16. Florian Stengel

    Department of Biology, University of Konstanz, Konstanz, Germany
    Competing interests
    The authors declare that no competing interests exist.
  17. Helmut Bergler

    Institute of Molecular Biosciences, University of Graz, Graz, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7724-309X
  18. Alan John Warren

    Cambridge Institute for Medical Research, Cambridge, United Kingdom
    For correspondence
    ajw1000@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9277-4553

Funding

Medical Research Council (MC_U105161083)

  • Alan John Warren

Bloodwise (12048)

  • Alan John Warren

Wellcome (108466/Z/15/Z)

  • Edwin Chen

German Science Foundation Emmy Noether Foundation (STE 2517/1-1)

  • Florian Stengel

Collaborative Research Center (969 Project A06)

  • Florian Stengel

Austrian Science Foundation FWF Grants (P26136)

  • Helmut Bergler

Austrian Science Foundation FWF Grants (P29451)

  • Helmut Bergler

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2019, Kargas et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,606
    views
  • 579
    downloads
  • 58
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Vasileios Kargas
  2. Pablo Castro-Hartmann
  3. Norberto Escudero-Urquijo
  4. Kyle Dent
  5. Christine Hilcenko
  6. Carolin Sailer
  7. Gertrude Zisser
  8. Maria J Marques-Carvalho
  9. Simone Pellegrino
  10. Leszek Wawiórka
  11. Stefan MV Freund
  12. Jane L Wagstaff
  13. Antonina Andreeva
  14. Alexandre Faille
  15. Edwin Chen
  16. Florian Stengel
  17. Helmut Bergler
  18. Alan John Warren
(2019)
Mechanism of completion of peptidyltransferase centre assembly in eukaryotes
eLife 8:e44904.
https://doi.org/10.7554/eLife.44904

Share this article

https://doi.org/10.7554/eLife.44904

Further reading

    1. Structural Biology and Molecular Biophysics
    Mia L Abramsson, Robin A Corey ... Michael Landreh
    Research Article

    Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

    1. Structural Biology and Molecular Biophysics
    Jesse Howe, Douglas Walker ... Elisar J Barbar
    Research Article

    53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.