From the journal RSC Chemical Biology Peer review history

29-Apr-2021

Dear Prof Liu:

Manuscript ID: CB-ART-04-2021-000079
TITLE: <b>Deciphering the substrate recognition mechanisms of the heparan sulfate 3-<i>O</i>-sulfotransferase-3 </b>

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Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology

************

Reviewer 1

Wander et al present an analysis of the substrate recognition mechanisms used by the HS sulfotransferase HS3ST3. HS3ST3 is an enzyme of high biomedical interest, both due to the importance of HS, and the particular role of this enzyme in generating the sulfation pattern exploited by HSV1 for cellular entry. The same group previously reported the basis of substrate recognition for HS3ST1, a related enzyme responsible for generating the HS pattern involved in antithrombin binding. This latest work complements those previous results, allowing us to better understand the distinct molecular mechanisms used by two 3O-sulfotransferases to discriminate sequences in HS to create unique sulfation patterns.

This is a well-writen manuscript of a well-executed project, which presents a compelling rationale for why HS3ST3 prefers 6O unsulfated substrates for activity, namely the propensity for 6O-sulfated substrates to bind in a nonproductive mode to the enzyme. Although most of the manuscript was compelling, I must admit I did find the final mutational analyses to be somewhat unsatisfying, as they do not really address the relative contributions of unproductive vs productive substrate binding in solution. I appreciate that a full treatment of this problem would be a significant undertaking beyond the scope of a simple revision. However, there is one experiment that I would like to see carried out.

- Mutation of R190, which is involved in a key bidentate interaction in the unproductive binding mode is a good idea in principal. The authors find that mutation to Ala dramatically reduces enzymatic activity against both oligos 1 and 3. However, R190K may be a more appropriate mutation to test, as this should remove the bidentate interaction involved in unproductive binding of 3, whilst maintaining the charge and H-bonding capabilities that may be important for productive binding. It would be good if this mutant was made and subjected to the same activity analysis as the others.


Minor points
- P9 “In the superposition of 3-OST-1 and 3-OST-3, both the catalytic glutamates of 3-OST-1 (Glu90) and 3-OST-3 (Glu184) and the acceptor 3-OH from the two substrates superimpose well with the 3-OH groups located 5.6 Å and 6.0 Å from the PAP 5- phosphorous atoms, respectively.” A comma is needed between “…superimpose well” and “with the 3-OH…”, otherwise the meaning of sentence changes.
- P13 “…to the greater loss in activity of K190A…” Should this be R190 or K259?
- Fig 5D, bottom sugar. The arrow pointing to the acceptor site is not pointing to O3 of sugar c’.
- Ref 21 page number should be 5265 rather than 5256
- The crystallographic statistics in table 1 are fine, although there is one oddity, at least to my eye. The I/sI values suggest that the data processing could have perhaps gone to slightly higher resolution. However, the outer shell data completeness appears low, especially for the 8-mer 1 complex. Was there a particular issue with data processing? If so, perhaps mention it in the methods.
- Table 2 – footnote “The 100% activity of…was determined to be the transfer of XX pmoles of sulfo groups”. In what timeframe? I gather 1 h from the methods? Please include this info in the footnote.
- Suppl. Fig S6 – Since a main feature of the nonproductive binding orientation is that it is ‘backwards’ compared to the productive orientation, it seems more appropriate that the bottom panel here is also drawn backwards?
- Suppl reference 5 – Weird formatting for the volume number? (60 pt 12 Pt 1)

Reviewer 2

This manuscript addresses a very challenging problem - understanding the molecular interaction of charged oligosaccharide to large GAG binding sites of enzymes. The generation of suitable substrates alone is very impressive. Mechanistic studies here are supported by structural studies which provide a unique insight into the biological recognition events. The authors clearly identify two binding modes, one productive and one unproductive, which is very interesting. The experiments are well executed and the manuscript is well presented.

May 18, 2021

Dr. Gonçalo Bernardes
Associate Editor
RSC Chemical Biology

Re: Manuscript ID: CB-ART-04-2021-000079

Dear Dr. Bernardes,

We are submitting a revised manuscript for publication as a full paper in RSC Chemical Biology. We were very pleased with the reviewers’ comments on the importance and quality of our work. The manuscript has been revised in order to address all comments and questions raised by the reviewers. The altered material in the manuscript is highlighted in yellow.


Referee #1

1. Wander et al present an analysis of the substrate recognition mechanisms used by the HS sulfotransferase HS3ST3. HS3ST3 is an enzyme of high biomedical interest, both due to the importance of HS, and the particular role of this enzyme in generating the sulfation pattern exploited by HSV1 for cellular entry. The same group previously reported the basis of substrate recognition for HS3ST1, a related enzyme responsible for generating the HS pattern involved in antithrombin binding. This latest work complements those previous results, allowing us to better understand the distinct molecular mechanisms used by two 3O-sulfotransferases to discriminate sequences in HS to create unique sulfation patterns.

This is a well-written manuscript of a well-executed project, which presents a compelling rationale for why HS3ST3 prefers 6O unsulfated substrates for activity, namely the propensity for 6O-sulfated substrates to bind in a nonproductive mode to the enzyme.

We are thankful for the comments about the importance and quality of our work.

2. Although most of the manuscript was compelling, I must admit I did find the final mutational analyses to be somewhat unsatisfying, as they do not really address the relative contributions of unproductive vs productive substrate binding in solution. I appreciate that a full treatment of this problem would be a significant undertaking beyond the scope of a simple revision. However, there is one experiment that I would like to see carried out.

- Mutation of R190, which is involved in a key bidentate interaction in the unproductive binding mode is a good idea in principal. The authors find that mutation to Ala dramatically reduces enzymatic activity against both oligos 1 and 3. However, R190K may be a more appropriate mutation to test, as this should remove the bidentate interaction involved in unproductive binding of 3, whilst maintaining the charge and H-bonding capabilities that may be important for productive binding. It would be good if this mutant was made and subjected to the same activity analysis as the others.
We appreciate the suggestion for analysis of an additional enzyme mutant. The suggested experiment has been carried out and the results added to the summary table of mutant enzyme activity (Table 2). The R190K mutant enzyme displayed similar reactivity to 8-mer 1 and 8-mer 3 as the wild type. No further discussion of the R190K mutant was added to the revised text, as the conclusion remained the same.

3. P9 “In the superposition of 3-OST-1 and 3-OST-3, both the catalytic glutamates of 3-OST-1 (Glu90) and 3-OST-3 (Glu184) and the acceptor 3-OH from the two substrates superimpose well with the 3-OH groups located 5.6 Å and 6.0 Å from the PAP 5- phosphorous atoms, respectively.” A comma is needed between “…superimpose well” and “with the 3-OH…”, otherwise the meaning of sentence changes.

A comma has been added to the text at the indicated location.

4. P13 “…to the greater loss in activity of K190A…” Should this be R190 or K259?

The text has been corrected to R190A.

5. Fig 5D, bottom sugar. The arrow pointing to the acceptor site is not pointing to O3 of sugar c’.

The arrow has been adjusted to indicate the proper site for 3-O-sulfation.

6. Ref 21 page number should be 5265 rather than 5256

The reference has been corrected.

7. The crystallographic statistics in table 1 are fine, although there is one oddity, at least to my eye. The I/sI values suggest that the data processing could have perhaps gone to slightly higher resolution. However, the outer shell data completeness appears low, especially for the 8-mer 1 complex. Was there a particular issue with data processing? If so, perhaps mention it in the methods.

We did not experience any particular issues with data processing. The choice of resolution cutoff is always a little tricky as there is no set standard. We do not make our decisions from a single value such as I/sigI but rather by balancing a number of considerations, including redundancy, CC1/2, completeness, percentage of reflections with I/sigI > 2, etc. We particularly pay attention to the resolution choice based on these values in the last shell so we don’t overstate the effective resolution and quality of the structure. For the 8mer-1 data set extending the resolution to 2.3 Å dropped the completeness in the last shell to 58% which we deemed too low to report, so we stepped back to 2.34 Å where we were > 70% complete (77%).

8. Table 2 – footnote “The 100% activity of…was determined to be the transfer of XX pmoles of sulfo groups”. In what timeframe? I gather 1 h from the methods? Please include this info in the footnote.

The footnote has been edited to include the time length of the experiment (1 hour).

9. Suppl. Fig S6 – Since a main feature of the nonproductive binding orientation is that it is ‘backwards’ compared to the productive orientation, it seems more appropriate that the bottom panel here is also drawn backwards?

While we appreciate the suggestion, we originally attempted to draw the oligosaccharide in the “backward” orientation but found the resulting structure to be more confusing than helpful and so omitted it from the manuscript.

10. Suppl reference 5 – Weird formatting for the volume number? (60 pt 12 Pt 1)

The formatting has been changed.

Referee #2

11. This manuscript addresses a very challenging problem - understanding the molecular interaction of charged oligosaccharide to large GAG binding sites of enzymes. The generation of suitable substrates alone is very impressive. Mechanistic studies here are supported by structural studies which provide a unique insight into the biological recognition events. The authors clearly identify two binding modes, one productive and one unproductive, which is very interesting. The experiments are well executed and the manuscript is well presented.

We appreciate the comments about the quality and significance of our work.

We hope the revised manuscript is suitable for publication in RSC Chemical Biology. We look forward to hearing from you on the matter.

Sincerely,


Rylee J. Wander
PhD Candidate
Dr. Jian Liu Lab
Pharmaceutical Sciences
Division of Chemical Biology and Medicinal Chemistry
Eshelman School of Pharmacy
University of North Carolina at Chapel Hill


Jian Liu, PhD
John A. and Deborah S. McNeill, Jr. Distinguished Professor
Eshelman School of Pharmacy
University of North Carolina
Email: jian_liu@unc.edu
Tel: 919-843-6511


Lars Pedersen, PhD
Staff Scientist, Head Structure Function Group
National Institute of Environmental Health Sciences/NIH
Email:pederse2@niehs.nih.gov
Tel:984-287-3538

28-May-2021

Dear Prof Liu:

Manuscript ID: CB-ART-04-2021-000079.R1
TITLE: <b>Deciphering the substrate recognition mechanisms of the heparan sulfate 3-<i>O</i>-sulfotransferase-3 </b>

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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With best wishes,

Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology

Reviewer 1

This revised version of the manuscript addresses all of my previous comments. I am happy to recommend publication.

Reviewer 2

The authors have addressed all issue raised by the reviewers satisfactorily