Binding to nucleosome poises human SIRT6 for histone H3 deacetylation

  1. Ekaterina Smirnova
  2. Emmanuelle Bignon
  3. Patrick Schultz
  4. Gabor Papai
  5. Adam Ben Shem

  • Curated by eLife

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    This manuscript provides a useful reconstruction of the structure of the sirtuin-class histone deacetylase Sirt6 bound to a nucleosome based on cryo-EM observations, and additional characterization of the flexibility of the histone tails in the complex based on molecular dynamics simulations. While similar structures have recently been published elsewhere, this solid study supports the conclusions of those papers and also includes new insights into the potential dynamics of Sirt6 bound to a nucleosome, insights that help explain its substrate specificity.

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Abstract

Sirtuin 6 (SIRT6) is an NAD + -dependent histone H3 deacetylase that is prominently found associated with chromatin, attenuates transcriptionally active promoters and regulates DNA repair, metabolic homeostasis and lifespan. Unlike other sirtuins, it has low affinity to free histone tails but demonstrates strong binding to nucleosomes. It is poorly understood how SIRT6 docking on nucleosomes stimulates its histone deacetylation activity. Here, we present the structure of human SIRT6 bound to a nucleosome determined by cryogenic electron microscopy. The zinc finger domain of SIRT6 associates tightly with the acidic patch of the nucleosome through multiple arginine anchors. The Rossmann fold domain binds to the terminus of the looser DNA half of the nucleosome, detaching two turns of the DNA from the histone octamer and placing the NAD + binding pocket close to the DNA exit site. This domain shows flexibility with respect to the fixed zinc finger and moves with, but also relative to, the unwrapped DNA terminus. We apply molecular dynamics simulations of the histone tails in the nucleosome to show that in this mode of interaction, the active site of SIRT6 is perfectly poised to catalyze deacetylation of the H3 histone tail and that the partial unwrapping of the DNA allows even lysines close to the H3 core to reach the enzyme.

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  1. Version published to 10.7554/elife.87989.5 on eLife
  2. Version published to 10.7554/elife.87989 on eLife
  3. Version published to 10.7554/elife.87989.4 on eLife
  4. eLife

    Author Response

    The following is the authors’ response to the previous reviews.

    We thank the reviewers for their remarks. Please find our detailed answers bellow.

    1. The authors' continued refusal to acknowledge the other reports before the final sentence of the Discussion, which has been pointed out in two previous rounds of review as a major flaw, detracts from the manuscript significantly.

    We now acknowledge and discuss the other SIRT6-nucleosome reports in the introduction as requested by the reviewer.

    1. While some of the grammatical errors in previous versions have been corrected, many remain, especially in the Methods section

    We corrected the remaining grammatical errors.

    1. Multiple statements of fact not supported by data shown in this work continue to lack appropriate references.

    We added references where facts were not supported by our data.

  5. eLife

    eLife assessment

    This manuscript provides a useful reconstruction of the structure of the sirtuin-class histone deacetylase Sirt6 bound to a nucleosome based on cryo-EM observations, and additional characterization of the flexibility of the histone tails in the complex based on molecular dynamics simulations. While similar structures have recently been published elsewhere, this solid study supports the conclusions of those papers and also includes new insights into the potential dynamics of Sirt6 bound to a nucleosome, insights that help explain its substrate specificity.

  6. eLife

    Reviewer #1 (Public Review):

    Smirnova et al. present a cryo-EM structure of a nucleosome-SIRT6 complex to understand how the histone deacetylase SIRT6 deacetylates the N-terminal tail of histone H3. The authors obtained the structure at sub-4 Å resolution and can visualize how interactions between the nucleosome and SIRT6 position SIRT6 to allow for H3 tail deacetylation. Through additional conformational analysis of their cryo-EM data, they reveal that SIRT6 positioning is flexible on the nucleosome surface, and this could accommodate the targeting of certain H3 tail residues. This work is significant as it represents the visualization of a histone deacetylase on its native nucleosomal target and reveals how substrate specificity is achieved. Importantly, it should be noted that recently two additional structures of the nucleosome-SIRT6 complex were already published. Therefore, Smirnova et al. confirm and complement these previous findings. Additionally, Smirnova et al. expand our understanding of the structural flexibility of SIRT6 on the nucleosome and clarify that SIRT6 also shows histone deacetylase activity on H3K27Ac.

  7. eLife

    Reviewer #3 (Public Review):

    Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

  8. Version published to 10.7554/elife.87989.3 on eLife
  10. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    We are grateful to the reviewers for their remarks, which significantly improved the paper. We repeated the biochemical assay concerning SIRT6 activity on H3-K27Ac and quantified the results as requested. Please find our detailed answers bellow each recommendation of the reviewers.

    Major recommendations:

    1. Grammatical errors are still common; the authors may need to consider an external editing service if they intend to fix the problems as they indicate that they believe the errors have been removed. The Results section is relatively clean, but parts of the Abstract, Introduction, and Discussion are more difficult to understand, and errors are especially common in the Methods section and those parts of the manuscript that are new in this revision.

    We corrected the grammatical errors.

    1. The introduction doesn't mention the other structures published; this is considered to be a serious deficiency as it prevents the reader from understanding the context for the contributions described here. Withholding the comparison with (or mention of) the previously published work to the last sentence of the Discussion seems misleading and does not give the reader adequate ability to judge the novelty of the results presented in this manuscript.

    A paragraph comparing our paper to the other structures published appear at the end of the discussion. We feel this is still the right place for such a paragraph.

    1. The addition of the assay for deacetylation is a significant improvement over the initial submission. This is important both for validating the importance of the acidic patch contacts and for helping to resolve the conflicting reports regarding activity on H3-K27Ac. Given the importance of this assay for the impact of the manuscript, it is not clear why the authors chose to 1) put the data in the supplement instead of in the main manuscript, and 2) provide only single samples without quantitation. These both seem to be significant limitations.

    We repeated the experiment and provided quantification of the results. We placed the figure in the main manuscript.

    1. The authors should add text or a table to the Methods section explaining which maps were used for each figure. By our count, there are 8 maps and 5 models (plus MD models) based on two datasets, but the relationships among them are not clearly stated, and the names of the maps (such as "Zn-finger focused" and "Rossman-Fold-Focused") might be changed to be more helpful to the reader (for example, the latter includes more than the Rossman fold and might be renamed "Sirt6-focused"). The authors should also explain how the maps were validated, which data were deposited in public repositories, and why some data were not deposited. For example, no statistics or methods regarding how particles were separated into integrated vs. non-integrated motion are provided for the CryoDRGN models. Further, the "two principle movements" described are depicted in 4 maps from two CryoDRGN runs using two separate sets of particles, but the relationships among them are not defined clearly. Finally, the connectivity of densities in Fig 8 are not obvious in the submitted maps. Until these points are addressed, the work is considered incomplete.

    AND

    1. The PDB model provided for review and submitted to the PDB database shows loosely bound DNA at the nucleosomal entry/exit points near the binding site of SIRT6, but the maps provided for review and submitted to the EMDB show stronger density for the canonical location of the DNA expected at these sites. The CryoDRGN maps support a more extended conformation, but these maps were not deposited or provided for review so their validity cannot be assessed.

    We added a section to the methods listing the different maps used for the figures. We deposited the map we used to trance the H2A N-terminal tail (EMD-18497). Unfortunately, we couldn’t deposit the cryoDRGN maps as the deposition system either accepts composite maps, where the consensus should be deposited too or experimental maps, where the deposition of half maps are mandatory. Nevertheless, the cryoDRGN maps are available upon request. We also added a supplementary figure (Supplementary Fig 6) to show how the cryoDRGN analyses were performed.

    1. The orientation, angle and threshold used in Fig 1 make it difficult to see the multiple DNA orientations that are visible in the deposited consensus map. Examination of the map suggests that the DNA model submitted to PDB corresponds to a weaker DNA conformation than is present in the map where both DNA conformations are visible. The authors should consider modeling both conformations in their deposited model to provide a more complete, accurate representation of the data. It is concerning that a key conclusion of the manuscript is that the DNA conformation changes upon SIRT6 binding, but density for the canonical position is observable in Fig 8a.

    Figure 1 is showing the overall representation of the SIRT6 bound nucleosome structure. We show the DNA linker orientations in the subsequent figure. Figure 8 (now Figure 9) shows the rearrangement of the SIRT6 Rossmann fold domain not the DNA linker.

    1. Figure 4 needs a more complete legend, indicating that it is a hybrid of the consensus structure (one color) and the MD simulations (another color). In general, the colors used in the figure should be changed to make the main points more accessible.

    As there is a color code for the histones, changing colors might be confusing. The figure legend mentions that panels c, d and e are from MD simulations.

    Minor recommendations:

    1. Figures 2c, e, and f are not referenced in the text.

    We now referenced all figure panels in the text.

    1. Consider moving Supp. 5C to Fig. 2 as the models in that figure come from the CryoDRGN maps and not the consensus map.

    Supplemental Figure 5c show the DNA linker deviation upon SIRT6 binding from another angle. We prefer to keep it there.

    1.) Supp Fig 3 is labeled "ZnF-nucleosome" refinement, but this appears to come from Data Set #2 processing. The map might be labeled ZnF-nucleosome but then a mask should be shown that excludes the Rossman Fold. It is not clear if this is a focused refinement or just a 2.9 A map that was merged with the "Rossman-fold" map.

    We changed both supplemental figures accordingly.

    1. The orientation of Fig 2 b and e do not show the differences in these models as well as panels c and f. Panels b and e could be replaced with the 4 CryoDRGN maps.

    The models reflect the cryoDRGN maps and panels c and f were added to clarify the movement.

    1. The MD description should emphasize that the H3 tails are moving with respect to the active site, as it currently suggests the active site is moving.

    In the results and in the discussion section we mention that we observe new conformations of the H3 tail, not of the active site.

    1. The authors refer to the "flexibility of the Rossmann fold domain," but the Rossman Fold domain isn't flexible, the linkage to the ZnF is flexible. Perhaps "observed conformational space" or "dynamic Rossman-fold domain position" are meant.

    The text was changed accordingly.

    1. The H2A C-terminal tail present in Fig 1 (bottom right) and Figure 3e is not present in the model in Fig 4a,b.

    The H2A tails conformation was not resolved in the cryoDRGN maps so we didn’t model it.

    1. The crosslinking agent used is not specified.

    The crosslinking agent used is specified more clearly in the methods.

    1. Supp Table 1 and EM methods do not agree on the magnification for Dataset #1. Verify nominal versus binned magnification and reported pixel size.
      The magnification in the methods was changed.
    1. Fig 3F showing the difference between affinity for H2A and H2A.Z-containing nucleosomes would be more convincing with a titration rather than the current comparison of a single concentration.

    We agree with this remark however, we find single concentration comparison is convincing enough for the purposes of this paper as it is not a central finding.

    1. Fig S1 legend; both the Zn-finger and helix bundle are stated to be shown in green.

    Figure S1 legend was changed.

  11. eLife

    eLife assessment

    This manuscript provides a useful reconstruction of the structure of the sirtuin-class histone deacetylase Sirt6 bound to a nucleosome based on cryo-EM observations, and additional characterization of the flexibility of the histone tails in the complex based on molecular dynamics simulations. While similar structures have recently been published, this solid study supports the conclusions of those papers and also includes new insights into the potential dynamics of Sirt6 bound to a nucleosome, insights that help explain its substrate specificity. Unfortunately, the authors do not mention the other recent publications until the end of their Discussion, and therefore provide little opportunity for comparison or context for the results presented.

  12. eLife

    Reviewer #1 (Public Review):

    Smirnova et al. present a cryo-EM structure of a nucleosome-SIRT6 complex to understand how the histone deacetylase SIRT6 deacetylates the N-terminal tail of histone H3. The authors obtained the structure at sub-4 Å resolution and can visualize how interactions between the nucleosome and SIRT6 position SIRT6 to allow for H3 tail deacetylation. Through additional conformational analysis of their cryo-EM data, they reveal that SIRT6 positioning is flexible on the nucleosome surface, and this could accommodate the targeting of certain H3 tail residues. This work is significant as it represents the visualization of a histone deacetylase on its native nucleosomal target and reveals how substrate specificity is achieved. Importantly, it should be noted that recently two additional structures of the nucleosome-SIRT6 complex were already published. Therefore, Smirnova et al. confirm and complement these previous findings. Additionally, Smirnova et al. expand our understanding of the structural flexibility of SIRT6 on the nucleosome and clarify that SIRT6 also shows histone deacetylase activity on H3K27Ac.

  13. eLife

    Reviewer #2 (Public Review):

    Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

  14. Version published to 10.1101/2023.03.20.533444v4 on bioRxiv
  15. Version published to 10.1101/2023.03.20.533444v3 on bioRxiv
  16. Version published to 10.7554/elife.87989.2 on eLife
  17. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    We are grateful to the reviewers for their remarks which significantly improved the paper. Following these remarks we completed the analysis and validation of our cryo-EM data and peformed several biochemical tests to support our conclusions, lending credbility to the paper. Please find our detailed answers bellow each recommendation of the reviewers.

    Major recommendations

    1. Errors and omissions in the presentation make the manuscript difficult to access.

    a) The text should be edited for grammatical errors more carefully

    • We corrected the grammatical errors.

    b) Figures should be labeled to allow the reader to follow the logic of the presentation and identify the features being discussed. Identification through the color coding (the identity of the histones, the location of zinc fingers, the active site, and so on) would be helpful.

    • We labeled the Rossman fold and Zn-finger domains in Figure 1 and described the histone color codes. The active site of SIRT6 is depicted in Figure 4.
    1. The recent publications from the Farnung/Cole and Peterson/Tan/Armache labs need to be cited and the results from Smirnova et al. compared and contrasted with those publications explicitly.
    • We added the following paragraph to the discussion section:
      “While this manuscript was under review two studies describing the structure of SIRT6-NCP appeared in press (Wang et al., 2023 ; Chio et al., 2023). The conclusion of these papers regarding the position of SIRT6 on the nucleosome and the unwinding of DNA by the enzyme are similar to our findings. We however dissected in addition the movements of SIRT6 on the nucleosome and analyzed via molecular dynamics the conformations of the H3 tail with respect to the SIRT6 active site. Our results point to the importance of the flexibility between the globular domains of SIRT6 and also explain how SIRT6 can access lysines that are much closer to the histone core than H3K9.”

    a) Notably, the Peterson/Tan/Armache labs suggest that H3K27 cannot be deacetylated by SIRT6 whereas the Farnung/Cole labs show deacetylation of H3K27 by SIRT6. Do the results of the Smirnova et al. structure help to resolve this situation?

    • We performed deacetylation tests of H3K27Ac nucleosomes and show that SIRT6 deacetylate H3K27Ac albeit at somewhat lower efficiency than H3K9Ac. Our molecular dynamics simulations explain how H3K27, which is close to the histone core, can still be reached by SIRT6 active site. We added the following text to the paper: “To lend support to this claim we tested whether SIRT6 can deacetylate residue H3K27 that was first acetylated by SAGA (Supplemental Fig. 7c). We find that indeed SIRT6 could efficiently deactylate H3K27Ac, although at a somewhat slower rate than H3K9Ac. We conclude that partial DNA unwrapping by SIRT6 allows H3-tail conformations that make lysines that are close to the core of H3 accessible to the enzyme.”

    b) The Farnung/Cole labs have visualized an intermediate state of deacetylation. How does this compare to the structure presented in this manuscript? Addressing these points would facilitate further research and discussion in the community.

    • We believe the resolution of the SIRT6 Rossmann fold precludes addressing these points.

    c) Can the authors exclude the possibility that the additional density observed in Supplemental Figure 6 is not coming from the H3 tail, as observed in the two other structures?

    • One density is the continuation of the H2A histone tail. We strongly believe that this density corresponds to this tail. The other density indeed can originate from the H3 tail. Therefore, we didn’t model anything inside it.

    d) It would be useful to comment on how much flexibility has been observed in the other structures for the SIRT6 interaction with the acidic patch, and also how other acidic-patch binding proteins compare with the results here.

    • We refrain from estimating the flexibility observed in the other structures as no such analysis is provided by these papers. Regarding the interaction with the acidic patch we mention that R175 packs against H2B L103 and serves as a classical “arginine anchor motif” and refer the reader to a review on the topic.

    e) Does the presence or absence of NAD+ affect the comparisons among the structures?

    • NAD+ binding might affect the fine structure of the active site although NAD+ was not observed in crystal stuctures of SIRT6 in its presence. The resolution of this part precludes further addressing this issue.
    1. The lack of biochemical validation of conclusions should be acknowledged and the reasoning behind this choice discussed.
    • We added experiments to validate our conclusions with biochemical tests. We produced nucleosomes with acetylatexd histone H3 by employing purified SAGA acetyltransferase complex. We isolated SIRT6 where the four residues implicated in interactions with the acidic patch are mutated to alanines (SIRT6-4A). We show that this mutant has very weak interaction with the nucleosome and much lower H3K9Ac deacetylation activity than WT. Similarly SIRT6-3A with mutations in the residues we suggest involved in binding to nucleosomal DNA also shows weak activity and binding to the nucleosome. We added Supplement Figure 7 that depicts the results of these experiments and embedded reference to these results in the approporiate sections of the text. Furthermore, we also show that SIRT6 is active in deacetylating H3K27Ac. This supports our molecular dynamics simulations showing that when SIRT6 binds the nucleosome, H3 tail can assume conformations where H3K27 is accessible by the enzyme’s active site. These results also appear in Supplement Figure 7.
    1. The authors nicely analyze and discuss the conformational flexibility of SIRT6 binding. This is an interesting finding, but Fig. 2 does not adequately convey this flexibility.
    • We now considerably improved Figure 2. We added panels c and f which depict clearly the movements we observe.
    1. The authors need to explain why two cryo-EM datasets were collected but were not merged, and the labeling of the datasets in the Supplemental Table appear to be switched.
    • The two datasets were collected with two very different pixel spacing therefore merging the two was possible only in Relion. This process, however, did not improve the resolution of the SIRT6’s Rossmann fold domain. We thank the reviewer to notice the discrepancy in the text and the Supplemental Table 1, it was corrected.
    1. Supplemental Figure 4 should be expanded to show additional representative densities with the respective fit of the model. This will allow the reader to better judge the quality of the data. At least the acidic patch interaction, the DNA-SIRT6 interactions, and the H2A should be shown in this context.
    • To illustrate the high-resolution features of the structure as well as the key regions we added Supplemental Figure 4.
    1. Standard elements of data analysis and validation should be included (angular distribution plots for cryo-EM reconstructions, a 3D FSC sphericity plot, a Q-score and EMRinger score for the cryo-EM data and atomic model, a model-to-map FSC curve). In general, model building is poorly described as it is unclear which maps (or to what degree different maps) were used for this process. This should be clarified in the methods section and in the Supplemental Table 1.
    • The model validation and data analysis details were added to Supplemental Figures 2 and 3 as well as in Supplemental Table 1.
    1. The provided maps also do not fully recapitulate the path of the H2A tail. The various density maps and PDB provided for this review do not support the final modeled residues of H2A between residues #118/119-123. This affects the validity of figure 3E and the discussion of the proximity of the potential substrates to the active site. The authors should clarify how they inferred that this is the H2A tail rather than the loosely bound SIRT6 Nterminal loop (whose stability could be altered by the presence or absence of NAD+) as suggested by overlaying the relevant crystal structures.
    • We added a panel to Supplemental Figure 4 (d) depicting the density where the H2A tail was modelled.
    1. The authors should explain how the data produced an asymmetrically oriented complex with a single SIRT6 molecule bound to one face. Were complexes with two SIRT6 molecules excluded? Is supplementary figure 4A the basis for the orientation and is this sufficient for this purpose?
    • Complexes with two SIRT6 molecules were present but only at around 1.5 percent of the whole dataset. These images were excluded from the refinement (shown in Supplementary Figure 2). The DNA orientation is depicted in Supplementary Figure 5A. The resolution obtained at the dyad (~2.5Å) allowed us to distinguish purine and pyrimidine bases. The Widom 601 sequence is asymmetric and the densities clearly show that there is only one orientation of the DNA observed with respect to SIRT6.
    1. The authors should clarify how supplemental figure 4B supports the conclusion that DNA is unwrapped. The density is not readily visible and docking of a simple DNA model in the ZN-focused map does not clearly rule out the possibility that this density comes from the H3 N-terminal tail.
    • We added to this figure the cryo-EM densities used to model the DNA path and the orientation of SIRT6. This image is now Supplemental Figure 5c.

    Minor recommendations

    1. The scale bar is missing for the 2D classes shown in Supplemental Figure 2.
    • We added the scale bar to the image depicting the 2D classes in Supplemental Figure 2.
    1. Masked classifications should be shown in the classification tree (Supplemental Figure 2 +3) with the masks shown as a transparent volume.
    • We now show the mask used for the 3D classifications of the SIRT6’s Rossman fold domain in Supplemental Figure 2.
    1. Supplemental Figure 3 should show the indicated 3D classifications in the classification tree.
    • We added the 3D classifications in Supplemental Figure 3.
    1. The authors should consider applying local CTF refinement and particle polishing to improve their resolution.
    • We did local and global CTF refinements. Polishing didn’t improve the resolution as movie frame alignment was done outside of Relion.
    1. The descriptions of the Widome 601 sequence orientation should be less ambiguous, perhaps mentioning the AT-rich and AT-poor arms instead of left and right arms.
    • We corrected the text as required.
    1. The statement "Such a large change in DNA trajectory is reminiscent of the chromatin-remodeler ATPases or pioneer transcription factors binding to nucleosome but was not observed in other histone modifiers" requires a citation.
    • We added approporiate references.
    1. The authors should provide a supplemental figure of the nucleosome-SIRT6 and PRC1-nucleosome structure comparison to complement the discussion section.
    • We refer the reader to the paper describing the PRC1-nucleosome structure.
  18. eLife

    eLife assessment

    This manuscript provides a useful reconstruction of the structure of the sirtuin-class histone deacetylase Sirt6 bound to a nucleosome based on cryo-EM observations, and additional characterization of the flexibility of the histone tails in the complex based on molecular dynamics simulations. Similar structures have recently been published, but this work provides solid support for the conclusions of those papers and also includes some novel insights into the potential dynamics of Sirt6 bound to a nucleosome that help explain its substrate specificity.

  19. eLife

    Joint Public Review:

    Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

    This manuscript is the third in a recent series of reports of cryo-EM structures of Sirt6:nucleosome complexes. The main conclusions of the three studies are similar, but this manuscript from Smirnova et al. includes additional molecular dynamics analysis of the histone tails. These studies suggest that part of the specificity for sites on the H3 tail is the result of only this tail having significant access to the active site. The results are partially validated by showing that H3-K27Ac is sometimes found near the active site in the simulations, and is a weak substrate for the deacetylase in vitro. All of the structures show Sirt6 contacting the acidic patch of H2A-H2B, partial displacement of the H2A C-terminal tail, and displacement of the DNA at the entry-exit site to "unclamp" the H3 N-terminal tail. This manuscript provides additional support for the conclusions drawn in the first two published structures, adds molecular dynamics simulations that provide further insight and includes a biochemical assay that helps to resolve an apparent conflict regarding the deacetylation of H3-K27Ac from the other two papers.

  20. Version published to 10.1101/2023.03.20.533444v2 on bioRxiv
  21. Version published to 10.7554/elife.87989.1 on eLife
  22. eLife

    eLife assessment

    This manuscript provides a useful reconstruction of the structure of the sirtuin-class histone deacetylase Sirt6 bound to a nucleosome based on cryo-EM observations, and additional characterization of the flexibility of Sirt6 based on molecular dynamics simulations. The analysis of the cryo-EM data is incomplete for some of the conclusions, and certain elements of the presentation were inadequate to allow sufficient evaluation. Biochemical validation of the conclusions is not provided, but some of this evidence has been published recently by two other groups in their analyses of the same complex. While much in this manuscript is confirmatory, the work also includes new insights into the potential dynamics of Sirt6 bound to a nucleosome.

  23. eLife

    Joint Public Review:

    Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. The cryo-EM analysis in its current form is incomplete, lacking aspects of validation such as angular distribution information and other standard measurements of the quality of the reconstruction. Biochemical validation of the structural findings is inadequate, relying primarily on previous publications. Importantly, two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes. This manuscript by Smirnova et al., therefore, confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

  24. Version published to 10.1101/2023.03.20.533444v1 on bioRxiv