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

Abstract
Data availability
Article and author information
Metrics

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

Philipp K Zuber

Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-5139-3930
Tina Daviter

The Institute of Cancer Research, London, United Kingdom

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2636-5959
Ramona Heißmann

Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany

Competing interests
The authors declare that no competing interests exist.
Ulrike Persau

Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany

Competing interests
The authors declare that no competing interests exist.
Kristian Schweimer

Biochemistry IV - Biophysical Chemistry, University of Bayreuth, Bayreuth, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3837-8442
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.

"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.

Copyright

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.