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  1. Author Response

    Reviewer #2 (Public Review):

    Zuber, et al. report structural and thermodynamic properties of 6 domains from the NusG superfamily of transcription factors, conserved in all kingdoms of life. This superfamily is characterized by an N-terminal NGN domain that binds RNA polymerase, affecting its activity. NGN domains are covalently linked to C-terminal domains (CTDs) that typically assume a single completely beta-sheet (KOW) fold. Recent work has shown, however, that one such domain, from E. coli RfaH, can switch from a completely alpha-helical fold into the all-beta-sheet KOW fold. Here, the authors identify a second fold-switching member of the NusG superfamily and investigate the physical basis of the dramatic switching transition by comparing thermodynamic and structural properties of fold-switching and single-folding CTDs.

    Strengths:

    To my knowledge, this is the first in-depth thermodynamic analysis of fold switching in the NusG protein family. One striking result is the stability difference between E. coli NusG (single-folding) and E. coli RfaH (fold-switching). It can be difficult to compare stabilities across organisms since their environments differ. For example, a fold-switching domain from a thermophile and single-folding domain from a mesophile might have similar stabilities. Clearer stability differences can be seen by comparing variants from the same species, which the authors show.

    The NMR experiments showing minor species in both fold-switching CTDs and one single-folding CTD suggest that the unfolded state plays an important role in fold switching. The 13C-alpha CEST experiments showing that the minor species E. coli RfaH CTD has helical character hints at a mechanism for how the RfaH CTD is poised to assume two different folds.

    Weaknesses:

    The thermodynamic and structural properties one single-fold domain (hSpt5-KOW) do not differ appreciably from a fold-switching domain, suggesting an incomplete mechanistic explanation of fold switching. Specifically, both the thermostabilities and the folding free energies of hSpt5-KOW (single-folding) and VcRfaH-KOW (fold-switching) were comparable. Furthermore, their 15N shift differences from CEST experiments (Figure 5 supplement 1B&C) appear similar. Thus, it is possible that the minor species of hSpt5-KOW has helical character like Ec- and VcRfaH. Furthermore, the secondary structure predictions showing hSpt5-KOW has largely beta-sheet propensities are suspect because the secondary structure predictions of MtNusG-KOW (single-folding) are inaccurate-they show helical propensities comparable to Ec- and VcRfaH (fold-switching, Figure 5 supplement 3). These propensities are not experimentally supported for MtNusG-KOW, indicating that predicted secondary structures are not always reliable.

    It is not clear why the authors state that the minor species of EcRfaH-KOW is in exchange between helical and completely unfolded conformations. The chemical shift differences in Figure 6A appear comparable, indicating one population.

    We agree that the chemical shift differences are similar (for both 15N and 13C). However, the increased 15N R2 values of the minor species indicate further exchange processes and together with the NMR-based chemical denaturation experiments our interpretation of this finding is that the minor species is an ensemble of largely unfolded species, some states of which are completely unfolded and some of which exhibit helical elements in regions 1 and 2. A detailed explanation is given in our reply to “Essential revisions #3”) and the manuscript has been modified to make clear which conclusions are experimentally proven and where we hypothesize.

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  2. Evaluation Summary:

    This is an interesting and timely paper that presents thermodynamic and structural (NMR) analyses of six KOW domains from the NusG superfamily of transcription factors. The authors identify a second fold-switching member of the NusG superfamily, VcRfaH, and investigate the physical basis of this fold-switching transition. The authors also compare the thermodynamic and structural properties of six fold-switching and single-folding KOW domains from different organisms, and show that fold-switching domains are less thermodynamically stable than their single-folding counterparts. This work will be of great interest for scientists in the fields of protein folding (theory and experiment), structural biophysics, and advanced protein NMR spectroscopy.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    This manuscript investigates the KOW domain of the fold switching, NusG protein family. E. coli RfaH-KOW is known to form an a-helical hairpin when docked onto the NGN domain and generate an auto-inhibited conformation, which blocks access to the RNA polymerase binding site. Upon activation, this helical KOW subdomain can refold into a beta-barrel structure that then is important for making contacts to ribosomes. In this manuscript the authors identify and structurally characterize RfaH protein from Vibrio cholerae, which has only ~36% sequence identity with EcRfaH, yet displays similar fold switching ability. In addition, the authors provide thermodynamic measurements and some structural information on 4 KOW domains from different organisms, and show that it is the instability in their β-barrel structure that enables the conformational plasticity and fold switching ability of these proteins.

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  4. Reviewer #2 (Public Review):

    Zuber, et al. report structural and thermodynamic properties of 6 domains from the NusG superfamily of transcription factors, conserved in all kingdoms of life. This superfamily is characterized by an N-terminal NGN domain that binds RNA polymerase, affecting its activity. NGN domains are covalently linked to C-terminal domains (CTDs) that typically assume a single completely beta-sheet (KOW) fold. Recent work has shown, however, that one such domain, from E. coli RfaH, can switch from a completely alpha-helical fold into the all-beta-sheet KOW fold. Here, the authors identify a second fold-switching member of the NusG superfamily and investigate the physical basis of the dramatic switching transition by comparing thermodynamic and structural properties of fold-switching and single-folding CTDs.

    Strengths:

    To my knowledge, this is the first in-depth thermodynamic analysis of fold switching in the NusG protein family. One striking result is the stability difference between E. coli NusG (single-folding) and E. coli RfaH (fold-switching). It can be difficult to compare stabilities across organisms since their environments differ. For example, a fold-switching domain from a thermophile and single-folding domain from a mesophile might have similar stabilities. Clearer stability differences can be seen by comparing variants from the same species, which the authors show.

    The NMR experiments showing minor species in both fold-switching CTDs and one single-folding CTD suggest that the unfolded state plays an important role in fold switching. The 13C-alpha CEST experiments showing that the minor species E. coli RfaH CTD has helical character hints at a mechanism for how the RfaH CTD is poised to assume two different folds.

    Weaknesses:

    The thermodynamic and structural properties one single-fold domain (hSpt5-KOW) do not differ appreciably from a fold-switching domain, suggesting an incomplete mechanistic explanation of fold switching. Specifically, both the thermostabilities and the folding free energies of hSpt5-KOW (single-folding) and VcRfaH-KOW (fold-switching) were comparable. Furthermore, their 15N shift differences from CEST experiments (Figure 5 supplement 1B&C) appear similar. Thus, it is possible that the minor species of hSpt5-KOW has helical character like Ec- and VcRfaH. Furthermore, the secondary structure predictions showing hSpt5-KOW has largely beta-sheet propensities are suspect because the secondary structure predictions of MtNusG-KOW (single-folding) are inaccurate-they show helical propensities comparable to Ec- and VcRfaH (fold-switching, Figure 5 supplement 3). These propensities are not experimentally supported for MtNusG-KOW, indicating that predicted secondary structures are not always reliable.

    It is not clear why the authors state that the minor species of EcRfaH-KOW is in exchange between helical and completely unfolded conformations. The chemical shift differences in Figure 6A appear comparable, indicating one population.

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  5. Reviewer #3 (Public Review):

    Zuber et al. investigated various KOW domains and found that the thermodynamically less stable EcRfaH and VcRfaH domains can switch between an all-alpha and all-beta state. This property has been known so far only for the KOW domain of the E. coli orthologue. For the latter, a very detailed thermodynamic and structural biology study could be performed at residue resolution by combining DSC and CD spectroscopy with very sophisticated NMR methods. The latter revealed the role of hydrogen bonds of the switching KOW domains by analyzing long range scalar couplings along hydrogen bonds. The second elegant approach was to use 15N and 13C CEST experiments to characterize the ensemble of conformations forming the unfolded state. This is a very difficult task to do experimentally. The authors can show experimentally at residue resolution that residues forming the alpha helices in the all-alpha switched form have already some alpha helical content in the U ensemble. Requirements for fold switching proteins from published theoretical approaches could be all experimentally confirmed and were convincingly discussed together with the Gibbs free energy landscape of all relevant conformational states. There is only one minor weakness concerning the interpretation of chemical shift changes of U with increasing urea concentrations (Figure 6B -Figure supplement 2).

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