Structural insights into the regulation of cohesion ... - The EMBO Journal

The EMBO Journal Peer Review Process File - EMBO-2012-83134

Manuscript EMBO-2012-83134

Structural insights into the regulation of cohesion establishment by Wpl1 Avradip Chatterjee, Silva Zakian, Xiao-Wen Hu and Martin R. Singleton Corresponding author: Martin R. Singleton, Cancer Research UK

Review timeline:

Submission date: Editorial Decision: Revision received: Accepted:

29 August 2012 02 October 2012 02 October 2012 10 January 2013

Editor: Hartmut Vodermaier Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision

02 October 2012

Thank you again for transferring your manuscript to The EMBO Journal, and please excuse the delay associated with its evaluation here. Following my own reading of the story and careful assessment of all the original reviewers' comments and your responses, I decided to send the manuscript to an expert arbitrating referee. This referee was asked to evaluate the manuscript from a structural biologists' point of view, as well as to arbitrate on the previous rounds of discussions between original referees and authors - and for this reason, I had made the original comments available to the reviewer, as well as your responses. Below you will find the comments of our arbitrating referee, numbered according to the original referees' (major) points in the second round of review. As you will see, our arbitrating referee is satisfied with a number of your responses on some of the key aspects, but s/he nevertheless also agrees that some of the original referees' criticisms would still appear valid and thus in need of further addressing before publication in a broad general journal such as The EMBO Journal. To achieve this, our referee recommends both alterations to the presentation and interpretation, as well as a few additional experiments (points 1.3 and 1.4). In conclusion, with the modifications and additional supporting data requested by our arbitrating referee, as well as the various minor changes as proposed in your response to the earlier comments, we would be willing consider the manuscript for publication in The EMBO Journal. I should however stress that for eventual acceptance, it will be essential to heed the arbitrator's recommendations, even for those aspects of interpretation where you may want to disagree with the more cautious views of the reviewer(s). Therefore, please make sure to provide a detailed point-bypoint response to the comments below with your final submission. When revising the manuscript for The EMBO Journal, please also carefully consider the following

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editorial points: - Please reformat the manuscript according to EMBO J guidelines, especially the referencing style. A more comprehensive Material and Methods section should be included in the main manuscript file, and PDB coordinates will need to be given at the stage of resubmission. - Please re-write the abstract so that it more explicitly summarizes key findings of the paper, rather than stating general background information that would be better placed in the Introduction. - Please also capitalize on our more extended article format to incorporate more of the directly relevant supplementary data into the main manuscript and main figures, and possibly also expand a bit more on the introduction and discussion sections. In particular, Supplementary Figures 2 and 3 could be moved into the main manuscript. Please also note that The EMBO Journal has no restrictions on the number of references, so you should be able to discuss all relevant background work, including most recent publications on the topic. - Finally, please include a brief Conflict of Interest statement at the end of the text. I should add that it is our policy to allow a single round of major revision only, and that competing manuscripts published during this period will have no negative impact on our final assessment of your revised study. I would appreciate if you could get back to me with an outlook on the requested modifications and your resubmission timeline, and of course please also let me know if you should have any additional questions regarding this decision and the revision requirements. Once again, thank you for the opportunity to consider this work! I look forward to hearing from you. _____ REFEREE REPORTS: Arbitrating Referee: Report based on reviews and responses. Reviewer 1. 1. The reviewer's comment is valid. Although it is reasonable for authors to engage in some speculation, there seems excessive speculation in this instance and it's a perfectly reasonable request that this speculative model is tested. Especially as this model is mentioned in the 'Results' section. The authors should either delete this entirely or tone it down and mention it in the Discussion. 2. I'm satisfied with the authors's argument. The region of the SMC3 peptide array corresponding to WIS1, WIS2 and WIS3 are the most reactive towards the Wpl1 probe. Reviewer 2 asks that the array data are described with more caution, and this would be appropriate. 3. The reduction in affinities of Wpl1-Smc3 interactions as a consequence of mutations either in WIS2 or in the motifs 3,4,5 and motifs 1 and motifs 2 are modest, i.e. 2-fold or 4-fold at best. This is surprising given that some mutations involve substitution of a buried Leu with an Asp. Thus, it is reasonable that the referees question the validity of the Wpl1-binding sites mapped onto Smc3, especially as the approach used (peptide array) is not very conventional and far from ideal given that Smc3 is a folded globular protein. Assuming that the authors have characterised correctly the important Wpl1-binding sites on Smc3, then a combination of mutations in all motifs would be expected to abolish Wpl1-Smc3 interactions. This is a testable hypothesis and should be done as a condition of manuscript acceptance. The mutated protein should be assessed for correct folding using CD. 4. In vivo experiments can be difficult to control for, and are less than an ideal method for testing mechanism (for some of the reasons the reviewer suggests). Nonetheless, the data can help refute or support a model. At best these data can be seen to support the model, assuming there are no artefacts due to miss-folding etc. Apart from showing that the mutants are correctly folded in vitro (i.e. CD which needs to be done), the authors have controlled for this experiment correctly and the data fits their model for Wpl1-SMC3 interactions. 5. These new data do distract. My own concern is that they are very weak. The mutant Wpl1 still


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associates with Pds5. No firm conclusions can be drawn from these data and these results should be removed from the manuscript. 6. This is a valid point. Acetylation does not abolish Wpl1-Smc3 interactions and therefore the association of Wpl1 to Smc3 is not acetylation dependent. To claim this would be to over-stating the case and to propagate a potentially erroneous mechanism and function for Sm3 acetylation. At most, the authors can only conclude the acetylation may weaken Wpl1-Sm3 interactions. The Discussion and Figure 6 should be revised. Reviewer 2 1. The query about the levels of the Wpl1 mutant (D272A, D273A). Although the author addressed this, he needs to make clear that the endogenous Wpl1 was tagged with an HA epitope. Then it is reasonable to accept that the mutant protein is expressed at the same level as wild type. 2. It does seem that the reviewer mis-understood the mutants used. The conclusions the author makes are valid (assuming the levels of Wpl1 are addressed). However this confusion draws attention to the need to improve clarity and organisation in this manuscript. 3. I agree with the reviewer that the authors exaggerate the data and there is insufficient evidence to claim that Wpl1 is the acetylation sensor. Crystallographic data. In general the crystal structure of the Wpl1 domain is of good quality (i.e. R-factors, stereochemistry etc.). However the electron density for the WIS2 peptide co-crystallised with Wp1 is not at all clear. The authors should show that a stereo view of this density and also a stereo view of simulated annealed 2Fo-Fc map after refinement of the complex. Other comments that need to be addressed. 1. Reviewer 1 questioned why the structure of WIS2 differs from the structure in SMC1. The author's response was not entirely convincing. They should include a figure comparing WIS2 as it is bound to Wpl1 and as it is in the context of Smc1. 2. In the in vivo experiment the authors mention that mutations of the WIS2 motif did not show a strong phenotype. The authors need to state 'data not shown'. 3. The manuscript suffers from over-speculation and lack of controls. For example page 6, lower paragraph is pure speculation and should be deleted. 4. Top line, page 11 'did not affect the folding of the protein'. How as this tested. 5. In many instances 'it's' should be 'its'.

Revision – authors’ response

02 October 2012

We are submitting our revised manuscript "Structural insights into the regulation of cohesion establishment by Wpl1". We have addressed the arbitrating reviewers comments as fully as we can. Please find details of our responses to individual points in the returned submission. We feel that these revisions, as well as those made in the previous round satisfactorily address the points raised. We have also followed your suggestion and moved supplemental figures 2 and 3 into the main manuscript, and revised the abstract and introduction to the paper. We hope you feel the manuscript is now suitable for publication in EMBO Journal.


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RESPONSES TO ARBITRATING REVIEWER REVIEWER 1 1. We have substantially revised the manuscript and tried to maintain a clearer separation between the experimental results and discussion. We have removed the speculation about the exact consequences of Eco1-mediated acetylation from this section, and substantially revised our discussion section to reflect the limitations of our experimental data. 2. We are pleased the referee accepts are argument with regard to the array data. We have modified the text to make clear that the peptide arrays represent an initial screen and require more detailed examination of the putative hits. 3. We have carried out the requested binding experiments with mutations in multiple sites and found that the combined mutations did not dissociate the complex, giving an overall affinity of approximately the same as the motif 2 mutation alone. While this was slightly contrary to our expectations, we should clarify that we were not intending to claim that we have fully characterised the interaction between Smc3 and Wpl1. Without a crystal structure of the intact complex, this seems to be rather difficult as we cannot make targeted mutations in the WIS1 and WIS3 interface that would be guaranteed to substantially disrupt binding. These are both rather large interfaces, so it is perhaps not surprising that one or two mutations in the each site do not abolish the interaction. In addition, it is not clear if there exists negative cooperativity between binding sites, or what the effect of conformational changes in Smc3 and Wpl1 (which are likely to occur) might have on the interactions. We do feel that this does not invalidate our results with individual interfaces, especially as the weakening of WIS1 and WIS3 correlates with in vivo phenotypes. The lack of phenotypes with WIS2 is a little harder to rationalise, but we feel that that total conservation of the WIS2 residues that interact with Wpl1, as well as the conservation of the Wpl1 binding site argues that this is a functionally important interaction. In addition, there are multiple precedents for other proteins binding the WIS2 helix in different systems. We do accept however, that the mode of binding and consequences of acetylation are likely to be more complex than we had originally proposed in the manuscript, and we have substantially toned down our interpretation of the results and been more explicit about the caveats in our discussion, and made the point that acetylation only weakens the WIS1 interface.

4. We have tested all our mutation proteins by thermal stability dye-binding assays (we did not have access to CD facilities within the timescale required for revision). Thermal stability assays have previously been employed to demonstrate correct folding (e.g. Chaugule at al., EMBO J. 30:2853) and work on the principle of a fluorescent dye binding to unfolded protein. As such, they are highly sensitive to destabilising mutations. The results (supplemental figure 4) show that all our mutants behave almost identically to the wild-type protein, and the gel filtration profiles (supplemental figure 6b) do not reveal the presence of any aggregation or other unusual properties. We therefore feel confident suggesting that the observed in in vivo phenotypes are most likely the result of reduced affinity for Smc3 resulting from a weaker binding by motifs 1 and 2. 5. We have removed the section on Pds5 interactions in its entirety. 6. We have revised the discussion to be more conservative in our claims, and have removed our models from figure 7.

REVIEWER 2 1. We have clarified our labelling of the Western blot to show that it is the endogenous WPL1 locus that has been tagged for the purposes of controlling for expression level. We have repeated the blot with a proper loading control, and show that the wild-type and mutant versions of Wpl1 re-introduced into cells are all expressed at approximately equivalent levels to the endogenous protein (figure 6d). 2. We have tried to clarify our description of the in vivo experiments. We hope that the revised figure 6d should help in this regard. 3. Please see response to point 6, reviewer 1. CRYSTALLOGRAPHIC DATA 1. We have included a stereo view of an OMIT map of the peptide (figure 5b) and that of the final refined 2Fo-Fc map (supplemental figure 3a).

OTHER COMMENTS 1. We have included the requested comparison between the WIS2 peptide and the helix in Smc1 (figure 5d) and discussed the possible reasons for the differences with Smc3 in more detail in the results section. We do feel that the varying conformations or disorder of this helix observed in different ABC ATPase structures do indicate that it might not be expected to be exactly the same as in Smc1. We should also point out that the N-terminal section of the peptide that participates in interactions with the Wapl domain has substantially different amino acid composition to the equivalent stretch in Smc1, further indicating that it might adopt a rather different structure. 2. We have included this sentence in the results section. 3. We have tried to include as many controls as time allows. We believe that the revised manuscript represents a more circumspect interpretation of the data, and we have removed the more speculative sections, including the indicated paragraph. 4. We were making the point that the mutations were made on the basis of the structure and would not be predicted to adversely affect protein folding (i.e. were not buried, or participating in interactions important for folding). This has now been tested experimentally as well (see point 4 for reviewer 1). 5. We have changed this where appropriate.


Acceptance letter

10 January 2013

Thank you for submitting your revised manuscript for our consideration. It has now been seen once more by our arbitrating referee (see comments below), and I am happy to inform you that there are no further objections towards publication in The EMBO Journal. Before we will able to send you a formal letter of acceptance, I need to ask you regarding one remaining editorial issue, the inclusion of the PDB coordinates of the newly determined structures. To expedite this, you may simply send them to us in the body of an email, from which we can easily copy them into the manuscript text file. After that, we should then be able to swiftly proceed with formal acceptance and production of the manuscript. Thank you again for this contributation to our journal and congratulations on a successful publication. Please consider us again in the future for your most exciting work. ___________________________________ Referee #1 (Remarks to the Author) The authors have addressed my comments and the manuscript is suitable for publication.


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