Dear Dr Varadarajan,

Thank you very much for submitting your Research Article entitled 'Mechanistic insights into global suppressors of protein folding defects' to PLOS Genetics.

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Josep Casadesús

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PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This manuscript describes the suppressor effects of second mutations on the defect of functions of proteins by first mutation. The authors performed various experiments including equilibrium and kinetics analyses of protein folding and derived conclusions that the significant factor is a refolding rate, and the stability of the native structure of a protein is not important. The topic authors treated is interesting and significant in terms of the evolution of protein functions. Thus, I feel that this manuscript can be basically published but the following points should be reconsidered before publication.

I think that the less significance of stability of a native structure for suppressor effects is plausible. But, the significance of refolding rate for suppressor effects is difficult to understand. What process of refolding is significant? The authors should discuss this point. Of course, the definite factor of this phenomenon may be difficult to demonstrate at this stage, but at least the authors should speculate this point.

It looks like that the native state of a suppressor mutant is less stable but its refolding rate becomes large as shown in the results of TYEM-1 b lactamase. How these phenomena can be interpreted in terms of mutations? Which mutations make the native state unstable and which mutations stabilize the folding transition state? The authors should give some speculations. The similar analyses should be given for other proteins.

Minor point;

In page 16, line 407, should “Figure 7G” be “Figure 7D”?

In page 16, line 410, should “Figure 7G” be “Figure 7E”?

In page 18, line 467, the conformation of from residue 9 to 15 (9KRESRYR15) should be presented in a figure.

Reviewer #2: This manuscript describes a remarkably thorough characterization of global suppressors in CcdB, (a toxin involved in the maintenance of F-plasmid in Escherichia coli) together with three other well-studied proteins. CcdB (Controller of Cell Death protein B) is particularly well characterized using a protocol that selects for a population of inactive mutants and then selects for global suppressors within this population.

Mutations are identified that stabilize the WT protein but fail to act as global suppressors, and the results provide persuasive support for an overall conclusion that "thermodynamic stabilization is neither necessary nor sufficient for suppression." Rather, as summarized in the Discussion, "the findings collectively indicate the role of kinetic stability, in particular an increase in the refolding rate constant as being primarily responsible for the suppressor phenotype."

The authors are seeking an underlying mechanism that explains global suppression, an issue of fundamental importance. Indeed, the finding that enhancement of refolding rates in these suppressors is important, but I don't think it is a "mechanism" in the usual sense of the word. The attendant finding that inactivating mutants largely affect the core while global suppressors localize at the solvent-accessible surface (especially in loops) is both intriguing and suggestive.

What is it about distal loops that can affect global suppression? For what it's worth, an old result from John Gerlt comes to mind. Here, deletion of an entire loop in staphylococcal nuclease resulted in a protein that is significantly more active than wild type. [Poole et al (1991) Deletion of the omega-loop in the active site of staphylococcal nuclease. 1. Effect on catalysis and stability, Biochemistry 30, 3621-3627. Baldisseri et al (1991) Deletion of the omega-loop in the active site of staphylococcal nuclease. 2. Effects on protein structure and dynamics, Biochemistry 30, 3628-3633.]

A question that arises is the interaction between the CcdB loop that includes suppressor candidate residues 10-12, and cognate structures, such as CcdA. The authors do raise this issue, but I would have welcomed slightly more structural information about the extent to which protein:protein binding might complicate the interpretation of suppression. Along similar lines, the high resolution structures of S12G, V46L and S60E seem to invite detailed assessment of differences in hydrogen-bonding, solvent accessibility, and possible intramolecular interactions between proximal loops, beyond those shown pictorially in fig. 6.

To show I read the paper carefully:

lines 294, 296: fold, not "folds", e.g., 10-fold, not "10-folds".

line 488: To, not "Till"

line 502: delete final comma before the period.

This is an outstanding paper

Reviewer #3: In this work Varadarajan and coworkers explore biophysical mechanisms by which global suppressors act on several proteins whose knockout mutations they suppress.

The analysis focuses on two key biophysical factors – thermal stability and folding kinetics using several proteins from various sources and therapeutic areas as example. The authors find that while suppressor mutations often increase stability to compensate for destabilization by detrimental mutations this is not always the case and they note a number of exceptions. In terms of kinetics the authors see a more consistent trend whereby suppressor mutations speed up in vitro folding kinetics both in fast and slow phase and they attribute the rescue effect primarily to kinetic rather than thermodynamic stabilization though direct link between kinetic stabilization and functional effect of suppressors in vivo has not been established in this work. However in discussion they speculate that somehow kinetic stabilization might lead to increase in abundance of functional proteins in the cell, perhaps through the effect on cotranslational folding mediated by non-ATP dependent chaperones.

The paper is interesting and the analysis is careful and comprehensive and I recommend publication with enthusiasm after the following concerns are considered:

1) The authors emphasize that effect on kinetic rather than thermodynamic stability determine suppressor mutations. Figure 8 is supposed to convey this message but its presentation is cursory, even most notations are not defined and it doe not impress on the reader that thermodynamic effects of suppressors are less significant than kinetic ones. E.g. amplitudes a_0 and A_0 are not defined and presentation of Fig.8 appears only in the Discussion. I am not sure therefore how Fig.8 conveys main message of the paper.

2) In Fig.1 kinetic traces are provided but no analysis of rates, amplitudes etc are given

3) The authors suggest that the effect of mutations on vitro kinetics may indicate a more profound effect on cotranslational folding and resulting yields of functional proteins. Recent paper PMID: 31911473 provides evidence of direct link between kinetic traps manifest in biphasic folding kinetics in vitro and effects on cotranslational folding. In this sense it may be worthwhile to explore the linear association between PIM and/or loci of rescue mutations and slow (for E. coli) codons in endogenous Ec genes such as ccdB

4) The authors point out to examples of stabilizing rescue mutations. However, one the most striking stabilizing mutations that gives rise to evolution of antibiotic (TMP) resistance is L28R in Ec DHFR. A 2016 paper PMID: 26929328 provides a detailed biophysical analysis of this stabilizing rescue mutation and its epistatic effects on abundance, activity, antibiotic binding etc and possible tradeoffs including effects of chaperones and proteases.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes: Takeshi Kikuchi

Reviewer #2: Yes: George Rose

Reviewer #3: No

Dear Dr Varadarajan,

We are pleased to inform you that your manuscript entitled "Mechanistic insights into global suppressors of protein folding defects" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Associate Editor

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Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

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Comments from the reviewers (if applicable):

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