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 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 form might drive the global folding transition in metamorphic proteins in general.

Fluid protein fold space and its implications

“…Chemical exchange saturation transfer (CEST) experiments demonstrated that another fold‐switching protein, the C‐terminal domain of RfaH (discussed in the following section), populates its folded state at 95% and a partially folded state with residual secondary structure corresponding to the fold‐switched state at 5%. [ 59 ] In both cases, the folded ensembles of these proteins populate both distinctly folded states, explaining how environmental triggers can switch the proteins from one energetically favorable conformation to the other.…”

“…[ 98 ] Although PTMs have not been shown to switch a protein's structure from one stable fold to another, an observation of an intrinsically disordered protein (IDP) suggests that such transitions may be possible for fold‐switching proteins, which tend to have less stable ground states than canonical globular proteins. [ 31,59 ] Specifically, extensive NMR analysis revealed that multi‐site phosphorylation induces the neural IDP 4E‐BP2 to fold. [ 99 ] This study demonstrates that post‐translational modifications can dramatically shift the relative populations within a protein's structural ensemble.…”

“…Recent work has shown that the ensemble of the fold‐switching domain of RfaH is poised to assume both α‐helix and β‐sheet structures. [ 59 ] Additionally, a handful of mutations can shift the relative balance between two conformations of another fold‐switching protein, XCL1. [ 37 ] Based on these observations, it seems plausible for one or several post‐translational modifications to shift the equilibrium of a protein capable of populating multiple folds from one dominant state to another (Figure 3).…”

“…[ 119,120 ] Furthermore, recent NMR results demonstrate that the ensemble of conformations accessible to a fold‐switching protein might poise it to assume folds with disparate secondary structures, while similar secondary structure propensities were not detected in the ensemble of one of its single‐folding homologs. [ 59 ] Nevertheless, the general relationship between the conformational heterogeneity of a protein's ensemble and its fold‐switching propensity is not well understood. Furthermore, little is known about the relative energies of fold‐switching conformations [ 41,59 ] or the mechanisms of fold‐switching transitions.…”

“…[ 59 ] Nevertheless, the general relationship between the conformational heterogeneity of a protein's ensemble and its fold‐switching propensity is not well understood. Furthermore, little is known about the relative energies of fold‐switching conformations [ 41,59 ] or the mechanisms of fold‐switching transitions. [ 55,59,105,121 ] Additional studies could uncover important mechanistic details of fold switching.…”