From the journal RSC Chemical Biology Peer review history

Berlin, October 21, 2020

Dear Dr Conibear:

Manuscript ID: CB-ART-10-2020-000175
TITLE: Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

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Technische Universität Berlin
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Reviewer 1

High mobility group (HMG) family N (HMGN) proteins are involved in a wide range of biological processes by interacting with nucleosome core particles and modulating chromatin architecture and conformational dynamics. Posttranslational modifications (PTMs) of HMGN proteins are biologically important and likely to regulate the binding between HMGN proteins and the nucleosome. In this paper, the authors install site-specific PTMs by protein semisynthesis coupled with solid phase peptide synthesis and use segmental isotope labeling to study functional and structural roles of acetylation and phosphorylation on HMGN1 protein mutants. It is non-trivial to obtain sufficient materials after many chemical and biochemical steps with good purity. The authors utilize the unique power of NMR spectroscopy to illustrate conformational perturbations caused by the N- and C-terminal PTMs on the intrinsically disordered HMGN1. Electrophoretic gel mobility binding assays were also performed to assess the functional role of the PTMs on HMGN1. Overall, this manuscript is well written; especially, the method is clearly documented with sufficient detail. I have the following minor comments:

(1). There are two unassigned peaks labeled with * in Figure 2e. What are they?
(2). It would be informative to supplement the secondary chemical shift (SCS) of C_alpha in Figure 2f using the SCS of C_beta and C’.
(3). The authors may consider numbering the HMGN1 sequence in Figure 3a to help readers map the chemical shift perturbation in Figure 3d to its sequence. The same may apply to Figure 4a.
(4). It is interesting to see some long-range chemical shift perturbation in HMGN1 with pS6. Have the authors considered adding some salt to see if such perturbation would be reduced by Debye charge screening? As there is salt in the electrophoretic gel assay, the presence of salt may reduce the perturbation caused by pS6 or other C-terminal PTMs on the binding of the nucleosome.
(5) The authors discuss the possibility of a peptidyl-prolyl isomerase, especially for pS85 that precedes a proline. Is there a minor peak next to the pS85 amide with a low intensity in Figure 4c?

Reviewer 2

Conibear and colleagues report on the semi-synthesis of high mobility group nucleosome-binding protein (HMGN1). This protein plays an important role in gene regulation and DNA repair. The protein is decorated by posttranslational modifications (PTMs) at the N-and C-termini, but the functions of these PTMs are still poorly understood. The authors established a native chemical ligation (NCL) scheme for installing K2ac and S6ph modifications at the HMGN1 N-terminus and expressed protein ligation (EPL) for introducing S85ph, S88ph, and S98ph into the C-terminal region. Radical desulfurization converted the obligate Cys residue to Ala, restoring the native HMGN1 sequence at both ligation sites. The central recombinant fraction was further labeled with stable isotopes, allowing subsequent NMR analysis of the modified proteins. NMR experiment uncovered a long-range effect of S6ph on residues 14-24, which are part of the nucleosome binding site. Follow-up NMR experiments with modified and unmodified HMGN1 peptides showed expected chemical shifts of modified residues but no new NOESY peaks as expect for major structural rearrangements. The C-terminal phosphorylation sites induced peak broadening, indicating the detection of multiple induced conformations. Finally, electromobility shift ways were performed in order to study a potential impact of the PTMs on nucleosome binding of HMGN1, but showed only little effects of these modifications.
The topic of this manuscript is clearly in the field of chemical biology and represents a very interesting example how chemical biology methods are applied for biophysical investigations of protein function and activity. Although a direct effect of HMGN1 modification on nucleosomal interactions could not be observed, conformational perturbations in specific regions of HMGN1 were observed, which could aid future investigations into the regulation of HMGN1 by PTMs. However, there are a few issues the authors should:

1.) When describing the results of the electrophoretic mobility shift assays the authors mention: “The semi-synthetic variants (HMGN1_unmodN and HMGN1_unmodC) showed similar binding to recombinantly produced HMGN1 (Fig. 5a), agreeing with the NMR data in showing that the semi-synthesis protocol does not change the structure or function of the protein.” I am not sure if I can follow this argument. Figure 5a shows that fully recombinant HMGN1 induces a quantitative shift of the nucleosome band at 50 nM to the 2x HMGN1-bound state. The corresponding band at 50 nM HMGN1 unmodC is very faint but only observed at the unbound level. This is also observed for HMGM1 unmodC (50 nM) in Fig 5c. The HMGN1 unmodN band in 5a is also not fully shifted to the 2x HMGN1-bound state. This issue complicates the interpretation of the binding data of the modified semi-synthetic HMGN1 proteins and should to be resolved.

2.) Labels of NMR signals in Figures 2e, 3b, and 4b are hardly readable du to intensive overlap, especially in the crowded regions of the spectra. The authors should consider revising these illustrations.

3.) The electrophoretic mobility shift assays in Fig. 5 indicate formation of bands beyond the 2x HMGN1-bound state at 200 nM with recombinant HMGN1. Is this nucleosomal disassembly or formation aggregates? Information about this observation would be helpful in the figure legend.

4.) In order to demonstrate the quality of the reconstituted nucleosomes, a picture of the full-scale native PAGE could be provided in the supporting information.

RSC Chemical Biology manuscript CB-ART-10-2020-000175
Dear Prof. Dr. Süssmuth,
Thank you for your initial evaluation and for the reviewers’ comments on our manuscript ‘Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications’ (Manuscript CB-ART-10-2020-000175). We would like to thank you and the reviewers for the insightful comments and suggestions and have revised the manuscript accordingly. We believe that these changes have strengthened this work and hope that with the changes and additions the manuscript is now suitable for publication in the RSC Chemical Biology.
The responses and changes are detailed below and highlighted in the revised manuscript and Supporting Information. All authors have approved the submission of this revised manuscript and responses to the reviewers’ comments. We have added a new author, Yasmin Dijkwel, who carried out the nucleosome binding experiments suggested by Reviewer 2. The original authors have all agreed to this addition. Please do not hesitate to contact me if you require any further information.
Yours sincerely,
Anne Conibear

Responses to reviewers’ comments
Reviewer 1
High mobility group (HMG) family N (HMGN) proteins are involved in a wide range of biological processes by interacting with nucleosome core particles and modulating chromatin architecture and conformational dynamics. Posttranslational modifications (PTMs) of HMGN proteins are biologically important and likely to regulate the binding between HMGN proteins and the nucleosome. In this paper, the authors install site-specific PTMs by protein semisynthesis coupled with solid phase peptide synthesis and use segmental isotope labeling to study functional and structural roles of acetylation and phosphorylation on HMGN1 protein mutants. It is non-trivial to obtain sufficient materials after many chemical and biochemical steps with good purity. The authors utilize the unique power of NMR spectroscopy to illustrate conformational perturbations caused by the N- and C-terminal PTMs on the intrinsically disordered HMGN1. Electrophoretic gel mobility binding assays were also performed to assess the functional role of the PTMs on HMGN1. Overall, this manuscript is well written; especially, the method is clearly documented with sufficient detail. I have the following minor comments:

Comment 1: There are two unassigned peaks labeled with * in Figure 2e. What are they?

Response: There are four low intensity peaks in the spectrum in Figure 2e that could not be conclusively linked to the rest of the sequence or to each other. They have now been marked as a, b, c and d in Figure S6.1 and their amino acid types noted in the figure legend. As none of these peaks is present in the spectra of the HMGN1 variants with residues 1-64 labelled, they seem to be from the 65-99 segment and we speculate that they are minor conformations of residues 75-79, as they are also lower in intensity than the other peaks. A comment has been added to Supplementary Figure S6.1 to explain this.

Comment 2: It would be informative to supplement the secondary chemical shift (SCS) of C_alpha in Figure 2f using the SCS of C_beta and C’.

Response: The secondary chemical shifts of Cβ and C′ have been calculated and are now shown in Supplementary Figures 8.2 and 8.3, respectively. This has been noted in the legend for Figure 2f. A sentence has been added on page 15 noting that the secondary shifts are < 1 ppm and support the conclusion from the Cα secondary shifts that there are no defined regions of secondary structure in HMGN1.

Comment 3: The authors may consider numbering the HMGN1 sequence in Figure 3a to help readers map the chemical shift perturbation in Figure 3d to its sequence. The same may apply to Figure 4a.

Response: As suggested by the reviewer, the sequences in Figures 3a and 4a have been numbered.

Comment 4: It is interesting to see some long-range chemical shift perturbation in HMGN1 with pS6. Have the authors considered adding some salt to see if such perturbation would be reduced by Debye charge screening? As there is salt in the electrophoretic gel assay, the presence of salt may reduce the perturbation caused by pS6 or other C-terminal PTMs on the binding of the nucleosome.

Response: We thank the reviewer for this helpful suggestion and have recorded 1H-15N HSQC spectra of all the HMGN1 variants with acK2 and pS6 modifications in 25 mM NaCl/ 25 mM KCl, corresponding to the salt concentrations used in the electrophoretic gel assays. A figure corresponding to Figure 3b, but with 25 mM NaCl and 25 mM KCl has been included in the Supplementary Data (Figure S6.3) and shows that the same residues shift as in the low-salt buffer, but the shifts tend to be smaller. This is better observed for the less-crowded spectra of the synthetic peptide variants of HMGN1_1-26 and HMGN1_65-99. To investigate whether salts in the buffer could screen electrostatic interactions of the posttranslational modifications, 1H-15N HSQC spectra of HMGN1_1-26 and HMGN1_1-26_acK2,pS6 were also acquired in NMR buffer, NMR buffer + 25 mM NaCl and 25 mM KCl, and in NMR buffer + 100 mM NaCl and 100 mM KCl, as shown in the revised Figure 3c. In this N-terminal HMGN1 segment, the changes in chemical shift between the modified and unmodified variants decrease slightly with increasing salt concentration, indicating a decrease in electrostatic interactions. For the C-terminal HMGN1 segments HMGN1_65-99 and HMGN1_65-99_pS85,88,98 (revised Figure 4c), the changes in chemical shift between modified and unmodified variants increase slightly with increasing salt concentration. Both results support our previous conclusions; posttranslational modifications of HMGN1 cause structural perturbations in residues distant from the modification site. The differences between the N- and C-terminal modifications might be rationalised by the different electrostatic contexts of the phosphoserine modifications; whereas phosphorylation of Ser6 introduces negative charge into an overall positively-charged region, phosphorylation of Ser85, 88 and 98 introduce negative charge into an already highly negatively charged region, likely increasing repulsion and chain extension. These NMR experiments with added salt have been added to the methods (page 12), figures 3c and 4c have been revised with the new data and a new Supplementary Data figure (Figures S6.3) has been added. The results are discussed in new sections highlighted on pages 20 and 26.

Comment 5: The authors discuss the possibility of a peptidyl-prolyl isomerase, especially for pS85 that precedes a proline. Is there a minor peak next to the pS85 amide with a low intensity in Figure 4c?

Response: In the spectrum of HMGN1_1-26, there is a very low intensity 1H-15N crosspeak at 8.45/115.6 ppm that might originate from a minor cis-Pro population. This peak is closest to the amide resonance of Ser88, but is too weak to assign and is not visible in the spectrum of the modified variant HMGN1_1-26_acK2,pS6. Although we cannot rule out cis-/trans-Pro isomerism as an explanation for the chemical shift changes we observe, our results for the short model peptides (Supp Fig 6.8), spectra in the presence of salt (revised Figs 3c and 4c) and the distance of the residues that shift from the PTMs suggest that electrostatic interactions are a more likely explanation. We have included this statement and rationale on page 26.

Reviewer 2
Conibear and colleagues report on the semi-synthesis of high mobility group nucleosome-binding protein (HMGN1). This protein plays an important role in gene regulation and DNA repair. The protein is decorated by posttranslational modifications (PTMs) at the N-and C-termini, but the functions of these PTMs are still poorly understood. The authors established a native chemical ligation (NCL) scheme for installing K2ac and S6ph modifications at the HMGN1 N-terminus and expressed protein ligation (EPL) for introducing S85ph, S88ph, and S98ph into the C-terminal region. Radical desulfurization converted the obligate Cys residue to Ala, restoring the native HMGN1 sequence at both ligation sites. The central recombinant fraction was further labeled with stable isotopes, allowing subsequent NMR analysis of the modified proteins. NMR experiment uncovered a long-range effect of S6ph on residues 14-24, which are part of the nucleosome binding site. Follow-up NMR experiments with modified and unmodified HMGN1 peptides showed expected chemical shifts of modified residues but no new NOESY peaks as expect for major structural rearrangements. The C-terminal phosphorylation sites induced peak broadening, indicating the detection of multiple induced conformations. Finally, electromobility shift ways were performed in order to study a potential impact of the PTMs on nucleosome binding of HMGN1, but showed only little effects of these modifications.
The topic of this manuscript is clearly in the field of chemical biology and represents a very interesting example how chemical biology methods are applied for biophysical investigations of protein function and activity. Although a direct effect of HMGN1 modification on nucleosomal interactions could not be observed, conformational perturbations in specific regions of HMGN1 were observed, which could aid future investigations into the regulation of HMGN1 by PTMs. However, there are a few issues the authors should:

Comment 1: When describing the results of the electrophoretic mobility shift assays the authors mention: “The semi-synthetic variants (HMGN1_unmodN and HMGN1_unmodC) showed similar binding to recombinantly produced HMGN1 (Fig. 5a), agreeing with the NMR data in showing that the semi-synthesis protocol does not change the structure or function of the protein.” I am not sure if I can follow this argument. Figure 5a shows that fully recombinant HMGN1 induces a quantitative shift of the nucleosome band at 50 nM to the 2x HMGN1-bound state. The corresponding band at 50 nM HMGN1 unmodC is very faint but only observed at the unbound level. This is also observed for HMGM1 unmodC (50 nM) in Fig 5c. The HMGN1 unmodN band in 5a is also not fully shifted to the 2x HMGN1-bound state. This issue complicates the interpretation of the binding data of the modified semi-synthetic HMGN1 proteins and should to be resolved.

Response: We agree with the reviewer that the gel shown in the original version of Figure 5a complicated the interpretation as the bands for HMGN1 unmodC were very faint. The electrophoretic mobility shift assays for panels a and c in Figure 5 have now been repeated and the revised figure has now been included. The bands for the HMGN1 variants bearing C-terminal modifications are now much clearer. Panel 5a shows unbound and 1:1 binding of all three variants at 50 nM HMGN1 and a mixture of 1:1 and 2:1 binding at 100 nM. With the clearer bands in the revised version of Panel 5c, there appears to be a trend of improved binding with the C-terminal PTMs and that it is stronger when multiple PTMs are present. This has been noted on page 27. The data clearly show that the HMGN1 variants all bind to nucleosomes, however the electrophoretic mobility shift assays were not as sensitive as we had hoped in distinguishing differences between the variants. Nevertheless, the data still allow us to conclude that neither the N- or C-terminal PTMs on HMGN1 abolish binding to nucleosomes, and that the C-terminal PTMs might even increase binding. This agrees with the literature that the central region of HMGN1 is primarily responsible for interactions with the nucleosome.

Comment 2: Labels of NMR signals in Figures 2e, 3b, and 4b are hardly readable due to intensive overlap, especially in the crowded regions of the spectra. The authors should consider revising these illustrations.

Response: To increase the clarity, Figures 3b and 4b have been modified to show labels only for the residues that shift. Residue labels have been removed from Figure 2e to show the distribution of cross-peaks more clearly. Expanded images with all the residues labelled are now shown in the Supplementary Data (Figures S6.1, S6.2 and S6.5).

Comment 3: The electrophoretic mobility shift assays in Fig. 5 indicate formation of bands beyond the 2x HMGN1-bound state at 200 nM with recombinant HMGN1. Is this nucleosomal disassembly or formation aggregates? Information about this observation would be helpful in the figure legend.

Response: At HMGN1:nucleosome ratios > 2:1, there are indeed bands beyond the 2 x HMGN1-bound state. Nucleosomal disassembly would result in lower bands and free DNA, which were not observed, and large insoluble aggregates would be unlikely to run into the gel. Instead, distinct bands are observed suggesting that, at high concentrations, more than two HMGN1 molecules can bind per nucleosome. We have included a new figure in the Supplementary Data (Figure S9.3), which more clearly shows these bands at higher HMGN1:nucleosome ratios. Indeed, the number of HMGN1 molecules per nucleosome present in the nucleus has been debated in the literature, although the HMGN1 concentrations generally considered to be limiting in the nuclear context (references 15 and 27). We have included this comment and reference to Figure S9.3 on page 27.

Comment 4: In order to demonstrate the quality of the reconstituted nucleosomes, a picture of the full-scale native PAGE could be provided in the supporting information.

Response: A picture of the full-scale native PAGE after assembly of the nucleosomes is now included as Figure S9.4. This shows co-localisation of bands from AF488 labelled DNA with the Cy-3 labelled histone octamers as single sharp bands.

Additional changes
- References that have now been published have been updated.
- A Table of Contents figure and text has been added.
- An error (pS86 should have been pS85) in the labels in Figure S9.2 has been corrected.

Berlin, December 16, 2020


Dear Dr Conibear, dear Anne:

Manuscript ID: CB-ART-10-2020-000175.R1
TITLE: Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 1

All of our comments have been satisfactorily addressed

Reviewer 2

Conibear and colleagues have provided a revised version of the manuscript “Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications”. The revisions were based on comments by two reviewers and a point-by-point response to all comments has been submitted together with the revised manuscript. The authors were able to address all comments and included additional experimental date into the revisions which further improved the manuscript. The topic is well within the scope of RSC Chemical Biology and the revised manuscript appears suitable for publication.