1. Structural Biology and Molecular Biophysics

Structural basis of ribosomal peptide macrocyclization in plants

  1. Joel Haywood
  2. Jason W Schmidberger
  3. Amy M James
  4. Samuel G Nonis
  5. Kirill V Sukhoverkov
  6. Mikael Elias
  7. Charles S Bond
  8. Joshua S Mylne  Is a corresponding author
  1. The University of Western Australia, Australia
  2. University of Minnesota, United States
https://doi.org/10.7554/eLife.32955
Share this article
Cite this article
  1. Joel Haywood
  2. Jason W Schmidberger
  3. Amy M James
  4. Samuel G Nonis
  5. Kirill V Sukhoverkov
  6. Mikael Elias
  7. Charles S Bond
  8. Joshua S Mylne
(2018)
Structural basis of ribosomal peptide macrocyclization in plants
eLife 7:e32955.
https://doi.org/10.7554/eLife.32955
  2. Download BibTeX
  3. Download .RIS

Abstract

Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, Helianthus annuus. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.

Data availability

The following data sets were generated

Article and author information

Author details

  1. Joel Haywood

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  2. Jason W Schmidberger

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  3. Amy M James

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  4. Samuel G Nonis

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  5. Kirill V Sukhoverkov

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  6. Mikael Elias

    Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St Paul, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Charles S Bond

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    Competing interests
    The authors declare that no competing interests exist.

  8. Joshua S Mylne

    School of Molecular Sciences, The University of Western Australia, Perth, Australia
    For correspondence
    joshua.mylne@uwa.edu.au
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4957-6388

Funding

Australian Research Council (FT120100013)

  • Joshua S Mylne

Australian Research Council (DP160100107)

  • Joshua S Mylne

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

Reviewing Editor

  1. Charles S Craik, University of California, San Francisco, United States

Version history

  1. Received: October 19, 2017
  2. Accepted: January 26, 2018
  3. Accepted Manuscript published: January 31, 2018 (version 1)
  4. Version of Record published: March 2, 2018 (version 2)

Copyright

© 2018, Haywood 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

  • 3,103
    views
  • 504
    downloads
  • 49
    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. Joel Haywood
  2. Jason W Schmidberger
  3. Amy M James
  4. Samuel G Nonis
  5. Kirill V Sukhoverkov
  6. Mikael Elias
  7. Charles S Bond
  8. Joshua S Mylne
(2018)
Structural basis of ribosomal peptide macrocyclization in plants
eLife 7:e32955.
https://doi.org/10.7554/eLife.32955

Share this article

https://doi.org/10.7554/eLife.32955

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Special Issue: Allosteric regulation of kinase activity

    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics

    Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article Updated

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.