Unconventional structure and function of PHD domains from Additional Sex Combs-like proteins

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   Feb 28, 2025

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   Feb 28, 2025

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   Referee #2

   Evidence, reproducibility and clarity

   Critique

   In this manuscript, the authors examine the biochemistry of two protein domains that are, on the basis of sequence similarity, predicted to function autonomously as binders of histone H3 tails or methylated DNA. They present solid data to suggest that neither domain in fact has this function, but that they act as protein interaction domains that form a heterodimer mediated by the presence of a zinc ion (two ligands from each protein).

   In the first part of the Results, the authors note that ASXL PHD doesn't contain aromatics that are characteristic of methylated lysine binding. I would just note that they don't mention at … More

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   Referee #2

   Evidence, reproducibility and clarity

   Critique

   In this manuscript, the authors examine the biochemistry of two protein domains that are, on the basis of sequence similarity, predicted to function autonomously as binders of histone H3 tails or methylated DNA. They present solid data to suggest that neither domain in fact has this function, but that they act as protein interaction domains that form a heterodimer mediated by the presence of a zinc ion (two ligands from each protein).

   In the first part of the Results, the authors note that ASXL PHD doesn't contain aromatics that are characteristic of methylated lysine binding. I would just note that they don't mention at this point that some PHDs bind unmethylated H3 - and that aromatics are not required for that binding activity. The lack of H3K4me3-binding aromatics doesn't at all make a case the domain doesn't bind histones. The lack of the Ala1 binding residues does make this case, but that's separate...

   Anyway, they then go on to show convincingly by ITC that ASXL doesn't bind the N-terminal H3 tail - unmodified or methylated. They also show modified-H3 ELISA data that make the same point (though it would be nice to know what the points were on the single ELISA that exceeded 2 SDs, even if they weren't reproduced - especially given there is a lot of scatter in the ELISA). I note in passing that I don't think I could find a Supp table 1).

   The authors then use AF3 to show that what would typically be the N-terminal zinc-binding site is not well predicted by the software (and the site ends up being square planar), suggesting that something might be amiss. (They were also unable to obtain an experimental structure.) It would have been helpful to gain more insight into what led them to the conclusion that the protein forms a weak homodimer based on the NMR data. Typically, it can be challenging to determine by NMR whether a dimer is forming or if non-specific soluble aggregates or other factors are contributing to line broadening.

   Next, the authors show nicely that MBD5/6 - two proteins shown in a previous paper to form a complex with ASXL - are predicted by AF3 to dimerize with ASXL - and form an intermolecular zinc-binding module in doing so. This is a nice result and there are very few examples of this in the literature (eg the zinc hook formed by Rad50 proteins). They confirm the zinc-binding prediction biochemically. They also show an HSQC of the complex (both subunits 15N labelled) and they count what they say is roughly the right number of peaks. To me, the lineshapes in the HSQC look good and, as the authors say, there are no clearly disordered resigies. I do make some additional comments below about the NMR data - suggesting what I think would be some valuable follow-up experiments. Overall, this study is a nice piece of biochemistry that recognizes an anomaly in the classification of examples of not one, but two, domain types well-known in the field of epigenetics. Going further than that, they not only show that the domains are mis-annotated but also demonstrate what their real function is and put forward a very likely model for their structure.

   The work is a good combination of AF based computational prediction with corroborating biochemistry and the experiments look technically well done to me. It is definitely of publishable quality and represents an advance in our understanding both of the particular proteins that they have studied and of the quirkiness of protein structure in general - there is always a new wrinkle to be discovered. I would make a couple of comments and suggestions that I think could improve the manuscript. I also have a number of minor comments below.

   Regarding the NMR data, the HSQC of the heterodimer that they show has nice lineshapes, as I mentioned above. However, the spectrum looks a little curious and closer inspection makes me wonder whether we are actually looking at two or more species with related structures. Many of the peaks appear to have a second peak nearby and it looks to me as if there is a consistent intensity ratio between the two forms (maybe 3:1 or 4:1?). It would be beneficial to explore this further, as understanding this aspect more clearly could have important implications for their analysis. I think the overall conclusions would probably still hold, but there would be far fewer signals than expected, suggesting likely some sort of slow-intermediate conformational exchange process that is giving two signals for a chunk of the residues and giving no signals for some of the others. Some comparison with the HSQC of the PHD domain alone might be helpful here.

   Some simple backbone triple resonance experiments would also be very helpful. Not only would they allow assignments to be made - and therefore a comparison of predicted secondary structure with the AF3-predicted fold - but also would help confirm whether there are two conformers. Often in these cases, the Ca and Cb chemical shifts for an exchanging system are much more similar than the HN and 15N signals, and it is therefore often clear that two peaks are actually the same residue in two different conformations. ZZ exchange experiments could help too, though these can sometimes be challenging.

   Finally, it would be reassuring to see SEC-MALS data for the heterodimer. Given that the interaction is mediated by covalent bonds, I'd expect to see a dimer molecular weight. It would also be reassuring to see a nice-looking SEC peak - and it would be useful data to have as part of the interrogation of possible chemical exchange mentioned above.

   Specific points

   • Intro: A nucleosome wraps less than two turns of DNA
   • I'm not a fan of this sentence: "The quaternary structure of the nucleosome forces the N- and C-terminal tails from histone proteins to protrude for covalent chemical modification". Not clear to me that the nucleosome 'forces' the tails to protrude...
   • The authors state that "Attachment of ubiquitin to histone H2A at K119 limits gene expression" - but they don't give any context. Which genes are limited in their expression? Nearby ones? Ones on the same chromosome? Just the gene that has an H2A-Ub in a specific position?
   • No need for capital Z in zinc.
   • "After purification, the protein solution was concentrated to 42.5 uM". The authors would not know the protein concentration to three significant figures. They would be unlikely to know it to 2 figures, given the inherent uncertainty in protein concentration measurement.
   • I like that they show purification gels for their proteins - almost no one does...
   • The authors state that "The domain, however, proved too small and flexible to produce crystals". However, the authors don't (as far as I can see) have any data to support the notion that either of these was the reason that no suitable crystals were obtained. I bet there are plenty of large, well-ordered proteins that haven't been able to have their crystal structures determined...
   • Supp fig 3 - the authors could label N and C termini.
   • "The 1H15N HSQC spectra revealed the presence of about 95 backbone amide peaks, which is in agreement with the overall protein complex." The authors could tell us how many peaks are expected, to make the comparison more useful! (and it should be spectrum).
   • "and form a tight, stable protein complex". Too many adjectives... The data don't show that the complex is tight, nor really say anything about its stability (is the Tm 35 degrees or 95 degrees - can't really say). The data do show that the two proteins form a complex.
   • I'd say that 633 A2 buried surface area isn't 'large'. It's small by protein complex standards, I think. But still perfectly reasonable.
   • Figure S6 - would be good to label N and C termini.

   Significance

   In this manuscript, the authors examine the biochemistry of two protein domains that are, on the basis of sequence similarity, predicted to function autonomously as binders of histone H3 tails or methylated DNA. They present solid data to suggest that neither domain in fact has this function, but that they act as protein interaction domains that form a heterodimer mediated by the presence of a zinc ion (two ligands from each protein).

   I am a structural biologist and biochemist who has worked on zinc-binding domains - including PHD domains - on and off over 30 years.

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3. 

   Review Commons

   Feb 28, 2025

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   Referee #1

   Evidence, reproducibility and clarity

   Summary: The Polycomb Repressive-Deubiquitinase (PR-DUB) complex catalyzes histone H2AK119Ub deubiquitinylation, regulating gene expression and chromatin dynamics. It comprises the BAP1 deubiquitinylase and one of three ASXL proteins (ASXL1-3). ASXL proteins contain a highly conserved C-terminal Plant Homeodomain (PHD), which was proposed to recognize epigenetic marks on histone H3 tail and other proteins. Mutations and truncations in the PHD domain are frequently observed in cancer. The authors propose that the ASXL PHD domain does not target histone H3 PTM marks. They model the PHD domain using AlphaFold3 and identified a … More

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   Referee #1

   Evidence, reproducibility and clarity

   Summary: The Polycomb Repressive-Deubiquitinase (PR-DUB) complex catalyzes histone H2AK119Ub deubiquitinylation, regulating gene expression and chromatin dynamics. It comprises the BAP1 deubiquitinylase and one of three ASXL proteins (ASXL1-3). ASXL proteins contain a highly conserved C-terminal Plant Homeodomain (PHD), which was proposed to recognize epigenetic marks on histone H3 tail and other proteins. Mutations and truncations in the PHD domain are frequently observed in cancer. The authors propose that the ASXL PHD domain does not target histone H3 PTM marks. They model the PHD domain using AlphaFold3 and identified a non-canonical fold that can apparently chelate one Zinc ion only in vitro, instead of the two ions typically bound by PHD domains. They also investigated the methyl CpG-binding domain proteins MBD5 and MBD6, known to interact with the ASXL PHDs and found that the complexes contain a composite Zinc-binding site at the interface between the two proteins. While the overall concept is interesting, the data do not justify conclusions. The authors should also reefer properly to the citations

   Major comments:

   • Are the key conclusions convincing?

   No. The final model is not substantiated by a robust experimental system The conclusions about Zinc binding and that PHD of ASXLs does not bind histone tails are based on a rather weak experimental system. There is a need for structural evidence and validation with mutagenesis. Also, comparing the sequence of the ASXL PHD to ING2 is insufficient and the PhD might bind other known or unknown peptide sequences on histones. The authors can not state or imply, based on their data, that the ASXL PHD does not recognize histone H3 epigenetic modifications. The methods are not sensitive enough and other peptides with an apparent fold enrichment have not been considered. It is not adequate to compare the Zinc-binding assay ASXL2 (residues 1375-1435) and Asx (residues 1610-1668) PHD domains with the RING domain of cIAP1 (residues 551-618) and a GST-only control. Why not other PHD domains?

   • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

   Yes

   • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

   Need solid evidence through experimental structure validation The ASXL PHD forms a composite Zinc-binding site with MBD5 and MBD6 is not well developed. There is a need structural validation

   • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

   Yes

   • Are the data and the methods presented in such a way that they can be reproduced?

   Need to provide more technical details

   • Are the experiments adequately replicated and statistical analysis adequate?

   It is unclear whether the data are mostly technical replicates in the same experiment as opposed to independent experiments

   Minor comments:

   • Specific experimental issues that are easily addressable.

   Need to validate Zinc chelation and composite interface for Zinc binding with other methods

   • Are prior studies referenced appropriately?

   Largely No Examples: - Attachment of ubiquitin to histone H2A at K119 limits gene expression (Cao & Yan, 2012) - Ubiquitin is attached to H2AK119 by the Really Interesting New Gene (RING) E3 ubiquitin ligase Polycomb Repressive Complex 1 (PRC1, Cohen et al., 2020) and is removed by the PR-DUB (Reddington et al., 2020; Scheuermann et al., 2010) - The PR-DUB has regulatory functions in the cell cycle, cellular development and DNA damageresponse, and determines short-term changes to gene expression (reviewed in Di Croce &Helin, 2013; Mozgova & Hennig, 2015; Parreno & Martinez, 2022; Schuettengruber et al., 2017).

   • Are the text and figures clear and accurate?

   Yes

   • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

   Validate the conclusions with robust methods.

   Other issues

   • In the Abstract: Need to include MBD5, MBD6 in the initial statement A Plant Homeodomain (PHD) at the C-terminus of ASXL proteins is recurrently truncated in cancer, and was previously proposed to recognise epigenetic modifications on the N-terminal tail of histone H3.

   Referees cross-commenting

   This session contains comments from both Reviewers

   Rev 1

   I believe that the manuscript needs substantial improvement. This involves experiments and this would require at least 6 months.

   Rev 2

   I don't agree that the authors need to determine an experimental structure for this work to be publishable. I think that the methods used, as is, are sufficient to draw a conclusion about the likely zinc ligation geometry. A structure would of course be great, but is a 'next level' experiment.

   Rev 1

   Ok, for not doing the structure, but this is just part of several comments. They have to address very carefully the comments and better control the study overall.

   Significance

   Could be significant and new if adequately demonstrated. The study is preliminary Could be significant in the filed of biochemistry and epigenetics.

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4. 

   Version published to 10.1101/2024.12.08.627434v1 on bioRxiv

   Dec 9, 2024