Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function

  1. Christina S Moesslacher
  2. Elisabeth Auernig
  3. Jonathan Woodsmith
  4. Andreas Feichtner
  5. Evelyne Jany-Luig
  6. Stefanie Jehle
  7. Josephine M Worseck
  8. Christian L Heine
  9. Eduard Stefan
  10. Ulrich Stelzl

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Abstract

NF2 (moesin–ezrin–radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation-dependent protein interactions. NF2 variants in the F2–F3 subdomain and the α3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F2–F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in NF2 related-schwannomatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.

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  1. Version published to 10.26508/lsa.202302043
  2. Review Commons

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    Reply to the reviewers

    1. General Statements [optional]

    *All three referee reports are very supportive. The referees acknowledge the insight obtained for NF2 tumor suppressor function, and they state unanimously that the appeal to the broader audience stems from the systematic deep mutational scanning approach employed. *

    Indeed, the goal of the study was to utilize conformation dependent NF2 protein interaction partners as a read out in deep mutational scanning interaction perturbation, which lead to the identification of an novel region, the F3 part of the FERM domain, important for NF2 conformation. The referees recognized the advance of the study and provided constructive feedback with excellent opportunities to improve the manuscript.

    *The points raised by the referees relate to the deep scanning analyses which needs additional explanations. Referee 1 has several comments with respect to experimental details, which we will address through text revisions and addition of data. Referee 2 suggests to include the Y2H in full (as supplemental part) and asks for more methodological discussion. We plan to include the data and will provide a new advantage / disadvantage discussion section for the deep scanning results. This is in line with Referee 3 who similarly says that “the deep mutational scanning interaction perturbation assay … message is somewhat lost in the main text”. *

    2. Description of the planned revisions

    Referee 1:

    In his/her first point the referee asks about justification of the use of the kinase in the Y2H experiments. Here we will report in more detail which kinases were used, in fact it was a discovery that ABL2 in contrast to all others tested did promote the NF2-PIK3R3 interaction. However, in the manuscript we provide evidence that the kinase dependency does not necessarily relate to NF2 phosphorylation. Rather we find mutations that relieved the PIK3R3-NF2 interaction from the kinase dependency. We show that the kinase promotes the PIK3R3 dimerization. We will make this point more clear in text revisions.

    We want to address the minor points 1-3 and 6-8 through revisions in the text, as we feel confident that the points can be addressed through better explanations and more detail.

    *Point 4: *

    We have examined expression of the YFP construct and will include the data in the revision.

    *Point 5: *

    We will reexamine the fluorescence images and provide better resolution pictures. Depending on the data we have, this may include new data were we record the localization of the NF2 variants again at higher resolution.

    Referee 2:

    Point 1: The bait construct which is missing from the panel was tested, but is autoactive and therefore the result can not be included in the figure. This will be clearly stated in the manuscript.

    *Point 2: The methodological part of the paper is important, however we failed to provide a discussion on the deep scanning result and agree that a critical discussion of advantages and disadvantages is warranted. *

    Minor points 1,2,4,5,6,7,9,10,12,13,14,15 can be addressed in full through text revisions.

    Point 3: Data will be added to supplemental Figure S1, however as we mentioned in the main text, the Iso1N and Iso7N, when used as prey do not result any interactions.

    Point 8: Taking the suggestion of the referee on board, we will provide a new Supplemental figure showing all variants that did not change the interaction patterns.

    *Point 11: We will fix the inconsistencies in Figure 5. We will include Q147 in the overall structure, S265 is a surface residue providing little structural information. *

    Referee 3:

    We thank referees 3 for their time and effort providing an assessment as experts on the molecular and clinical aspects of NF2.

    In response to the comments, we will strengthen the deep mutational scanning message through a new critical discussion part and fix the mistakes pointed at in the text.

    *We agree with the referees that “The use of isoform 7 as a construct is helpful to locate protein binding regions, but its physiological relevance is unclear.” This is exactly the point, to use a non-tumor suppressive isoform as a construct contrasting the binding behavior of the canonical isoform 1. We tried to summarize the knowledge about the non-canonical isoforms in the introduction (page2 bottom to page 3 top paragraph) as well as in Supplemental Figure 1. Unfortunately literature information is sparse. *

    Finally, we will check carefully (again) whether we used isoform 1 numbering throughout the manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The authors developed a deep mutational scanning interaction perturbation technique, based on reverse yeast two-hybrid analysis, to identify important regions influencing conformation dependent protein function in NF2.

    They test the tumor suppressive NF2 isoform 1 and a shorter non-tumor suppressive isoform 7 (lacking exons 2 and 3 and containing exon 16 instead of 17) and find three interacting proteins, KDM1A, EMILIN1 and PIK3R3 (KDM1A and EMILIN1 have been identified previously). They map binding regions of these proteins using fragments of NF2 isoforms 1 and 7 and by large-scale interaction perturbation mutation scanning.

    Major comments:

    The main scientific advancement in the study is the development of the deep mutational scanning interaction perturbation assay, but this message is somewhat lost in the main text of the results.

    The relevance of the binding protein that did not bind isoform 1 is unclear (PIK3R3) and the relevance of characterising the binding domains for three proteins with an unknown function is not made clear. Were these the only binding partners identified in the yeast screen? The use of isoform 7 as a construct is helpful to locate protein binding regions, but its physiological relevance is unclear. Does it have known expression or a known function in human cells?

    Minor comments:

    Nomenclature should be updated in line with the new guidelines (i.e. NF2 vs neurofibromin)

    The two major isoforms are 1 and 2, differentiated by their C-terminal region (exon 17 or Exon 16). It would be helpful to describe protein binding regions using the amino acid numbering of the full-length transcripts throughout the manuscript, rather than using isoform 7 numbering in some sections.

    "Closeness", should perhaps be changed to closed-ness

    The significance of the RT4-D6P2T and HEI-193 cell lines should be explained/indicated in the text.

    PPI should be expanded at first use.

    Results are included in the context of previous studies, but it needs to be made clearer in some places which results were found in previous studies and which were identified in the current study.

    Specific recommendations

    1. 'NF2 (Neurofibromine 2, merlin)' -delewte 'neurofibromine' this has been deleted by HGNC
    2. 'Genetic mutations or deletion of NF2 cause neurofibromatosis type 2,' -Replace neurofibromatosis type 2 with NF2 related-schwannomatosis and cite Legius et al Genet Med 2022

    Referees cross-commenting

    I cannot see any changes to this manuscript. In particular the terms 'neurofibromine' and neurofibromatosis should be deleted

    Significance

    The authors developed a deep mutational scanning interaction perturbation technique, based on reverse yeast two-hybrid analysis, to identify important regions influencing conformation dependent protein function in NF2.

    They test the tumor suppressive NF2 isoform 1 and a shorter non-tumor suppressive isoform 7 (lacking exons 2 and 3 and containing exon 16 instead of 17) and find three interacting proteins, KDM1A, EMILIN1 and PIK3R3 (KDM1A and EMILIN1 have been identified previously). They map binding regions of these proteins using fragments of NF2 isoforms 1 and 7 and by large-scale interaction perturbation mutation scanning.

    The main scientific advancement in the study is the development of the deep mutational scanning interaction perturbation assay, but this message is somewhat lost in the main text of the results.

    Dr Smith and Professor Evans are experts on the molecular and clinical aspects of NF2

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this manuscript the authors describe the use of a reverse Y2H-based, systematic mutational analysis method to study effects on conformation-dependent interactions of the NF2 tumor suppressor protein. Using this approach, they identified regions important for NF2 protein interaction and homomer formation and correlated some of these with cellular proliferation and matched patterns of known disease mutations. Overall, this work provides useful insight into NF2 tumor suppressor function (by identifying amino acids critical for NF2 conformational regulation) while demonstrating the power of their mutational scanning approach.

    Major Comments

    1. Why does Figure 1b not include interaction results for the NF2-Iso1N fragment as bait? Did the authors test this?
    2. The authors state that the majority of retested interactions behaved like WT (i.e., could not be confirmed; page 7 last paragraph) and claim this may be due to a difference in sensitivity between the deep scanning screen and pair wise spot testing. This seems like a very vague justification for the differences between the two assays; also, it's not immediately clear to me that the high-throughput scanning assay would necessarily be more sensitive than the lower-throughput pairwise comparison assay. The authors should provide a bit more discussion on this and address the possibility of false positives in their deep mutational scanning assay.

    Minor Comments

    1. Page 4, Line 3 from the bottom - should ready 'three isoform-specific protein interaction partners' not 'partner'.
    2. In Figure 1b, the interaction of NF2-Iso7-ex17 with PIK3R3 in the absence of ABL3 suggests that the observed kinase-dependence interaction of the NF2-Iso7 form may actually not be solely due to PIK3R3 homodimerization driven by phosphorylation. The authors should make note of this possibility in the text.
    3. On page 5 the authors mention that Iso1N and Iso7N, when used as preys, did not interact with full-length NF2. I don't see this experiment in the figures, however.
    4. On Page 6 (first paragraph) the authors state that the PIK3R3 interaction was 'promoted through pY-dependent PIK3R3 homodimerization'. While this is a likely and reasonable conclusion, they haven't explicitly shown this, so they should be careful about making such a strong statement. I'd recommend saying 'likely promoted' or something similar instead.
    5. In Figure S2, the Iso7 / EMILIN1 interaction does not appear to be giving the expected result in the rY2H (i.e., there is strong growth under both Y2H and r2H conditions). The authors should comment on/acknowledge this.
    6. For the deep mutagenesis screen, why wasn't an ABL2 condition used for NF2-Iso1C (see Fig. S2b)?
    7. For KDM1A and EMILIN1 the authors ran mutagenesis screens with both active and kinase dead ABL2, yet results were pooled. Were any differences observed in the effects of mutations on interaction between the two kinase conditions?
    8. Why aren't the yeast plates shown for most of the unconfirmed interactions? These could still be included in the Supplementary Material.
    9. On page 7, under Assessing Single Site Mutations, the authors refer to the Q147E mutation and reference Figure 3. However, Figure 3 shows only a Q147A mutation. Q147A is also referred to elsewhere. Which is the correct mutation?
    10. Figure S3b shows the 20 mutations presented in Figure 3. The DMS row indicates that some of these did not produce perturbations in the DMS experiments. Perhaps I'm misunderstanding here, but weren't the 20 mutations shown (and 60 total mutations) selected based on activity in the DMS assay? Or did some of the ones selected correspond to mutations which not produce an effect? Please clarify in the text.
    11. Why was the S265 mutation not considered in the structural analysis (other than being shown in Figure 4a, it isn't discussed). Also, Q147 (in the F2 region) is discussed and shown in Figure 4b, but not shown in the larger overall structure in Figure 4a.
    12. The cell proliferation results are very difficult to meaningfully interpret. While it is clear that certain mutations do affect proliferation, consistency between different types of experiments and cell lines appears to be low.
    13. Perhaps a bit more discussion of the possible consequences of using yeast to study human NF2 interactions and how these might affect results would be useful (i.e., due to differences in membrane composition, cellular environment, post-translational modifications etc. between yeast and mammalian cells).
    14. Page 13, line 10 says 'your hypothesis'. Believe should read 'the hypothesis'.
    15. Page 13, line 15 refers to '15' NF2 variants showing altered PPI patterns; however, 16 were described in the manuscript.

    Significance

    This work provides insight into how NF2 conformational changes relate to tumor suppressor function, which is particularly valuable since this area is still not well understood and published results have sometimes appeared contradictory. In addition to the insights into NF2 biology provided, the manuscript also demonstrates the value of the deep scanning mutagenesis approach. Overall, the presented research is very solid and, assuming the comments presented above (most of which are minor) are addressed I have no trouble recommending it for publication.

    I believe that the NF2 biology section will be of interest to a more specialized audience, while the general demonstration of the utility of the deep scanning mutagenesis will have broader appeal.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript "Missense variant interaction scanning reveals a critical role of the FERM-F3 domain for tumor suppressor protein NF2 conformation and function" examines the effect of a reasonably exhaustive set of point mutations on the NF2 protein on protein-protein interactions and intra-protein interactions for two isoforms of NF2 (1 and 7), finding an interesting pattern of mutations in the region not associated with bindings nevertheless impact binding, and that this binding is sometimes dependent on the presence of kinase ABL2. Authors justify this by arguing conformation shifts in the protein, potentially regulated by phosphorylation, and with distinct conformations between isoforms 1 and 7 creating different interaction patterns, must explain the differences in binding properties. The paper specifically examines mutations to phosphomimetic (i.e., charged, so as to mimic phosphorylation) amino acid residues, with relevance for the probable biological regulation of this binding. Authors note that previous work has found inconsistent protein binding properties for phosphomimetic or phosphor-inhibiting substitutions on S518, in different conditions, which would be explained by other regulation of these conformational changes, a reasonable argument. Structural modeling of the mutants and their potential effects on a "closed" NF2 structure are intriguing and well-appreciated to support the paper's conclusions, and the paper is overall well-reasoned and convincing, and it should be published.

    Concerns:

    The kinase ABL2 is used to perturb NF2 phosphorylation, and this is not adequately justified. Kinases such as PAK2 (PMID: 11782491, PMID: 11719502) and PKA (PMID: 14981079) target NF2. In the methods referred to (Grossmann et al), nine tyrosine kinases were used for their screen, and while ABL2 was used in this paper and generated numerous interactions, it is not clear that ABL2 is the appropriate kinase to use here. The exhaustive use of many kinases would obviously be impractical and unreasonable for this study, but the choice of this kinase should be clearly explained.

    Minor issues:

    1. In the intro, authors write "While the other ERM protein family members do not have activities directly linked to cancer, NF2 tumor suppressor activity was initially characterized in flies and mice". While "directly" makes the statement technically true, it could be argued that ERM protein involvement is as legitimate as the tumor suppression activity of NF2 (PMID: 11092524, PMID: 24421310), and therefore the suggested contrast is slightly misleading. This has no relevance to the broader paper or its findings.
    2. Figure 2b: Authors state mutational coverage is fairly even across the protein, however there appears to be a notable spike around a.a. 180? This does not match any of the site mutations later found to be particularly relevant for interactions, which cluster around 250 and 450, and is therefore not a significant issue.
    3. In the methods, cell concentration is at one point said to be 'concentrated to an OD600 of 40-80'. I have never seen cell concentration expressed this way. Authors no doubt grew cells to an OD between 1 and 2 and concentrated ~40-fold as is standard, and wish perhaps to avoid estimating concentrations as cell numbers, which would only be approximate and cell size-dependent? However, OD is only linear between 1 and 2 for cell concentrations. An OD above 4 simply cannot be observed, as all light would be blocked. Methodology here is sound, this is merely an unusual way of expressing things.
    4. Page 10: The authors point out that they cannot see any difference in the expression levels of the NF2 mutants. However there is no quantification of the immunofluorescence signal supporting this information. Maybe a western blot could suffice this argument.
    5. It is very difficult to see the localization of NF2 mutants with the immunofluorescence images as they are very small. May be try with a 63X objective or focusing on just one or two cells or adding insets with higher magnification would allow the reader to view the details of Nf2 localization.
    6. 5th line from the bottom on page 8: allowed to model -> allowed us to model
    7. Line 8 from the top on page 12: inY2H -> in Y2H
    8. Line 10 from the top on page 13: your hypothesis -> our hypothesis

    Significance

    Dear Editor,

    The manuscript "Missense variant interaction scanning reveals a critical role of the FERM-F3 domain for tumor suppressor protein NF2 conformation and function" examines the effect of a reasonably exhaustive set of point mutations on the NF2 protein on protein-protein interactions and intra-protein interactions for two isoforms of NF2 (1 and 7), finding an interesting pattern of mutations in the region not associated with bindings nevertheless impact binding, and that this binding is sometimes dependent on the presence of kinase ABL2. Authors justify this by arguing conformation shifts in the protein, potentially regulated by phosphorylation, and with distinct conformations between isoforms 1 and 7 creating different interaction patterns, must explain the differences in binding properties. The paper specifically examines mutations to phosphomimetic (i.e., charged, so as to mimic phosphorylation) amino acid residues, with relevance for the probable biological regulation of this binding. Authors note that previous work has found inconsistent protein binding properties for phosphomimetic or phosphor-inhibiting substitutions on S518, in different conditions, which would be explained by other regulation of these conformational changes, a reasonable argument. Structural modeling of the mutants and their potential effects on a "closed" NF2 structure are intriguing and well-appreciated to support the paper's conclusions, and the paper is overall well-reasoned and convincing, and it should be published.

  6. Version published to 10.1101/2022.12.11.519953v2 on bioRxiv
  7. Version published to 10.1101/2022.12.11.519953v1 on bioRxiv