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

• Published in: eLife 2022-10, Vol.11
• Main Authors: Zuber, Philipp K, Daviter, Tina, Heißmann, Ramona, Persau, Ulrike, Schweimer, Kristian, Knauer, Stefan H
• Format: Article
• Language: English
• Subjects: Amino acids; Energy; fold-switching; KOW domains; metamorphic proteins; NMR; Nuclear magnetic resonance; NusG proteins; Physiology; Protein folding; Proteins; RfaH; RNA polymerase; Spectrum analysis; Structural Biology and Molecular Biophysics; Transcription factors
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.76630

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 Kyrpides, Ouzounis, Woese (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 toward 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.