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

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 alpha-hairpin and a beta-barrel. In contrast, NusG/Spt5-KOW domains only occur in the beta-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 beta-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 alpha-hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

Automated summary

This study investigates the thermodynamic stability and structural dynamics of two-domain protein RfaH and NusG/Spt5-KOW domains. The findings suggest that the folding transition in RfaH is driven by transiently structured elements in the unfolded conformation.