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

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 -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 towards the -hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.