1. Structural Biology and Molecular Biophysics

Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins

  1. Philipp K Zuber
  2. Tina Daviter
  3. Ramona Heißmann
  4. Ulrike Persau
  5. Kristian Schweimer
  6. Stefan H Knauer  Is a corresponding author
  1. University of Bayreuth, Germany
  2. The Institute of Cancer Research, United Kingdom
https://doi.org/10.7554/eLife.76630
Share this article
Cite this article
  1. Philipp K Zuber
  2. Tina Daviter
  3. Ramona Heißmann
  4. Ulrike Persau
  5. Kristian Schweimer
  6. Stefan H Knauer
(2022)
Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins
eLife 11:e76630.
https://doi.org/10.7554/eLife.76630
  2. Download BibTeX
  3. Download .RIS

Abstract

The two-domain protein RfaH, a paralog of the universally conserved NusG/Spt5 transcription factors, is regulated by autoinhibition coupled to the reversible conformational switch of its 60-residue C-terminal KOW domain between an α-hairpin and a β-barrel. In contrast, NusG/Spt5-KOW domains only occur in the β-barrel state. To understand the principles underlying the drastic fold switch in RfaH, we elucidated the thermodynamic stability and the structural dynamics of two RfaH- and four NusG/Spt5-KOW domains by combining biophysical and structural biology methods. We find that the RfaH-KOW β-barrel is thermodynamically less stable than that of most NusG/Spt5-KOWs and we show that it is in equilibrium with a globally unfolded species, which, strikingly, contains two helical regions that prime the transition towards the α-hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

Data availability

Coordinates for VcRfaH-KOW have been deposited to the Protein Databank (ID: 6TF4). Chemical shifts have been deposited in the Biological Magnetic Resonance Databank under the following accession numbers: #28039 (hSpt5-KOW5), #28040 (MjSpt5-KOW), #28041 (VcRfaH) and #34450 (VcRfaH-CTD). Source data files have been provided for Figures 2, 3, 5, and 6

Article and author information

Author details

  1. Philipp K Zuber

    Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5139-3930

  2. Tina Daviter

    The Institute of Cancer Research, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2636-5959

  3. Ramona Heißmann

    Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany
    Competing interests
    The authors declare that no competing interests exist.

  4. Ulrike Persau

    Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany
    Competing interests
    The authors declare that no competing interests exist.

  5. Kristian Schweimer

    Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3837-8442

  6. Stefan H Knauer

    Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany
    For correspondence
    stefan.knauer@uni-bayreuth.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4143-0694

Funding

European Cooperation in Science and Technology (CA15126)

  • Philipp K Zuber

The COST action funded PKZ's research stay to conduct DSC experiments at the Birkbeck UCL, UK.A DFG grant (to Paul Rösch, former head of department) funded PKZ's position and parts of the research material.

Reviewing Editor

  1. Rina Rosenzweig, Weizmann Institute of Science, Israel

Version history

  1. Received: December 23, 2021
  2. Preprint posted: January 15, 2022 (view preprint)
  3. Accepted: October 13, 2022
  4. Accepted Manuscript published: October 18, 2022 (version 1)
  5. Version of Record published: November 23, 2022 (version 2)

Copyright

© 2022, Zuber 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

  • 802
    views
  • 184
    downloads
  • 13
    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. Philipp K Zuber
  2. Tina Daviter
  3. Ramona Heißmann
  4. Ulrike Persau
  5. Kristian Schweimer
  6. Stefan H Knauer
(2022)
Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins
eLife 11:e76630.
https://doi.org/10.7554/eLife.76630

Share this article

https://doi.org/10.7554/eLife.76630

Categories and tags

Research organism

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.