From the journal RSC Chemical Biology Peer review history

23-May-2022

Dear Dr Hantschel:

Manuscript ID: CB-COM-04-2022-000108
TITLE: Synthesis of the Bcr-Abl SH2 domain for mirror-image monobody development

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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Reviewer 1

The Communication submitted by Schmidt et al. describes synthesis of the D- and L-versions of the SH2 domain of Bcr-Abl, which will be used for the development of D-protein binders. The 103-residue SH2 domain was synthesized in two segments, which were ligated using native chemical ligation followed by desulfurization at the ligation site to give the native sequence. To increase solubility and facilitate immobilization, an N-terminal poly-Lys spacer and biotin were incorporated. After solving some challenges in the synthesis, the ligation and desulfurization proceeded smoothly and the protein was refolded. Comparison with the recombinantly-expressed SH2 domain showed that structure and function were maintained in the synthetic proteins.
Overall, the communication is clearly and concisely written and was easy to follow. The data and methods are professionally presented and the results support the authors’ conclusions. Detailed methods are given in the supporting information and the referencing is well balanced. The very thorough methods described could be useful for others in the field and the synthetic proteins will be valuable tools for mirror-image monobody development. I would recommend acceptance of the communication in RSC Chemical Biology with consideration of the following minor revisions:
- A few more recent applications/examples of synthetic D-proteins could be mentioned and cited to show the broader applications of this strategy. (e.g. Weidmann_2019_CellChemBiol_26_645; Kent_2018_ProtSci_28_313)
- The sequence and numbering in Fig 1 are quite small and difficult to read.
- Page 2, paragraph 2: ‘For the generation of the N-terminal thioester …’ could be confusing as the thioester is on the C-terminus of the N-terminal segment. This should be re-worded to clarify.
- It might be helpful to include a sentence explaining that acetylation of the MeDbz linker would prevent formation of the N-acyl urea on reaction with p-nitrophenyl chloroformate and the base.
- Fig 5A – the recombinant SH2 domain seems to run slightly lower than the two synthetic proteins L-5 and D-5. Possible reasons for this could be mentioned. Similarly, the melting curve of recombinant SH2 shows a much sharper transition point (Fig S22), suggesting that the synthetic proteins might not be completely folded. Was a sample of the recombinant SH2 protein also denatured using the same protocol as for the synthetic proteins?

Reviewer 2

This manuscript written by Hantschel and co-workers describes chemical synthesis of Bcr-Abl SH2 domain that can be used for mirror-image screening. The synthesis using alanine-based NCL and desulfurization was straightforward, efficiently providing L- and D-Acr-Abl SH2 domains. The solubility problem of the N-terminal fragment was solved by introducing K5 residues and SG fragment to the N-terminus. The synthesized folding proteins were carefully identified by LC-MS, CD, and affinity to AS25, also comparing with the recombinant protein. Unfortunately, the mirror-image monobody screening (shown in the manuscript title) was not conducted. More importantly, the important contributions related to this study are not described. Thus, this manuscript can be accepted for publication in RSC Chemical Biology if the following major revisions are addressed:

(1) Mirror-image monobody, not actually developed in this study, is too much emphasized. This point should be mentioned as a future perspective.
(2) Important contributions on mirror-image screening are not adequately described. These must be properly introduced in the introduction section by citing the papers. For example, mirror-image phage display: Kim, Science 1996, 271, 1854; Cell 1999, 99, 103, and some related papers. Mirror-image screening of natural product library Oishi/Fujii, ChemComm 2016, 52, 7653; RSC Advances 2017, 7, 38725 (synthesis of D-Src-SH2 domain is included).
(3) Fig 1-4 are too small and not reader-friendly.

Dear Prof. Nikolovska-Coleska,
Thank you for the evaluation of our manuscript “Synthesis of the Bcr-Abl SH2 domain for mirror-image monobody development“ (CB-COM-04-2022-000108) to be considered for publication as a Communication in RSC Chemical Biology.
We have carefully addressed the reviewer’s comments, and based on their suggestions and advice, we provide a revised version. Our modifications are explained point by point below and highlighted in yellow in the main manuscript.

Referee #1
The Communication submitted by Schmidt et al. describes synthesis of the D- and L-versions of the SH2 domain of Bcr-Abl, which will be used for the development of D-protein binders. The 103-residue SH2 domain was synthesized in two segments, which were ligated using native chemical ligation followed by desulfurization at the ligation site to give the native sequence. To increase solubility and facilitate immobilization, an N-terminal poly-Lys spacer and biotin were incorporated. After solving some challenges in the synthesis, the ligation and desulfurization proceeded smoothly and the protein was refolded. Comparison with the recombinantly-expressed SH2 domain showed that structure and function were maintained in the synthetic proteins.
Overall, the communication is clearly and concisely written and was easy to follow. The data and methods are professionally presented and the results support the authors’ conclusions. Detailed methods are given in the supporting information and the referencing is well balanced. The very thorough methods described could be useful for others in the field and the synthetic proteins will be valuable tools for mirror-image monobody development. I would recommend acceptance of the communication in RSC Chemical Biology with consideration of the following minor revisions:

Authors response: We thank the referee for the very positive comments on our manuscript and are happy that our efforts in reporting methods thoroughly was appreciated.

1) A few more recent applications/examples of synthetic D-proteins could be mentioned and cited to show the broader applications of this strategy. (e.g. Weidmann_2019_CellChemBiol_26_645; Kent_2018_ProtSci_28_313)

Authors response: We have added the suggested references in the introduction (page 1, right column before Fig. 1).

2) The sequence and numbering in Fig 1 are quite small and difficult to read.

Authors response: The figure was quite small, indeed. We have changed the font and increased the size of the sequence for better visibility, and changed the display of the numbers. To increase size and improve readability of the figures (see also comment 3 of reviewer #2), we have merged Fig. 5-6 with increased font sizes.

3) Page 2, paragraph 2: ‘For the generation of the N-terminal thioester …’ could be confusing as the thioester is on the C-terminus of the N-terminal segment. This should be re-worded to clarify.

Authors response: We have changed the sentence as suggested to “For the generation of the C-terminal thioester of the N terminal segment 1, …” (page 2, left column, last paragraph).

4) It might be helpful to include a sentence explaining that acetylation of the MeDbz linker would prevent formation of the N-acyl urea on reaction with p-nitrophenyl chloroformate and the base.

Authors response: We thank the reviewer for the suggestion. Indeed, this information was missing in the manuscript and we have extended the sentence: “Besides, capping with acetic anhydride after every coupling step is generally not recommended with the MeDbz linker as it prevents the subsequent N-acyl-N′-methylacylurea (MeNbz) formation and NCL” (page 2, right column).

5) Fig 5A – the recombinant SH2 domain seems to run slightly lower than the two synthetic proteins L-5 and D-5. Possible reasons for this could be mentioned. Similarly, the melting curve of recombinant SH2 shows a much sharper transition point (Fig S22), suggesting that the synthetic proteins might not be completely folded. Was a sample of the recombinant SH2 protein also denatured using the same protocol as for the synthetic proteins?

Authors response: The different migration behaviour in the SDS-PAGE analysis as seen in Figure 5A is consistent with the additional biotinylation and KKKKKSG-linker of the synthetic protein, thereby resulting in a higher molecular weight. In addition, we thank the referee for the observation that the recombinant SH2 domain shows a sharper transition point in our nanoDSF measurements (Fig. S22). Indeed, we initially wondered about this point as well: all three proteins were measured in parallel with the same denaturation protocol. The sharpness of the transition point could have been also affected by the biotinylation and KKKKKSG-linker of the synthetic SH2 domains. Importantly, the synthetic proteins even have a mildly higher melting temperature than the recombinant domain. Clearly, both the CD spectroscopy experiment (Figure 5C), as well as the ITC binding studies (Figure 5D-F and S19-20) clearly show that the synthetic proteins are folded and bind pY peptide ligands as the recombinant protein.

Referee #2
This manuscript written by Hantschel and co-workers describes chemical synthesis of Bcr-Abl SH2 domain that can be used for mirror-image screening. The synthesis using alanine-based NCL and desulfurization was straightforward, efficiently providing L- and D-Acr-Abl SH2 domains. The solubility problem of the N-terminal fragment was solved by introducing K5 residues and SG fragment to the N-terminus. The synthesized folding proteins were carefully identified by LC-MS, CD, and affinity to AS25, also comparing with the recombinant protein. Unfortunately, the mirror-image monobody screening (shown in the manuscript title) was not conducted. More importantly, the important contributions related to this study are not described. Thus, this manuscript can be accepted for publication in RSC Chemical Biology if the following major revisions are addressed:

Authors response: We thank the referee for the insightful comments. We are sorry for the confusion. Our study forms the basis for mirror-image monobody screening, but this was not conducted in this manuscript. Therefore, we have changed the title to “Synthesis of the L- and D- SH2 domain of the leukaemia oncogene Bcr-Abl” to capture the essence of our study.

1) Mirror-image monobody, not actually developed in this study, is too much emphasized. This point should be mentioned as a future perspective.

Authors response: As mentioned above, we changed the title to avoid any misunderstanding. Furthermore, we have described in the manuscript text with more clarity that the selection of mirror-image monobodies will be done in the future. (page 2, left column, first paragraph, and abstract).

2) Important contributions on mirror-image screening are not adequately described. These must be properly introduced in the introduction section by citing the papers. For example, mirror-image phage display: Kim, Science 1996, 271, 1854; Cell 1999, 99, 103, and some related papers. Mirror-image screening of natural product library Oishi/Fujii, ChemComm 2016, 52, 7653; RSC Advances 2017, 7, 38725 (synthesis of D-Src-SH2 domain is included).

Authors response: We apologize for not describing contributions on mirror-image screening systems adequately and have added a section with citation of the suggested references in the manuscript. (page 1, right column before Fig. 1)

3) Fig 1-4 are too small and not reader-friendly.

Authors response: We agree with the mentioned figures being not entirely reader-friendly and tried to improve them within the limits of the given page restrictions. We increased the sequence font size of Fig. 1 and increased the text size of Fig. 2 and Fig. 4. The size of Fig. 3 was enlarged and Fig. 5-6 were merged with increased font sizes.

We sincerely thank the reviewers for their constructive criticism and their efforts to improve our initial manuscript. We believe that the changes described above address all the issues in the original submission, and that these corrections have resulted in an improved manuscript. We are confident that the described findings, methodology and thorough analyses in our manuscript will be helpful for many scientists working on the interface of protein chemistry and biochemistry.

Looking forward to hearing from you,
Yours Sincerely,

Prof. Oliver Hantschel

01-Jul-2022

Dear Dr Hantschel:

Manuscript ID: CB-COM-04-2022-000108.R1
TITLE: Synthesis of the L- and D-SH2 domain of the leukaemia oncogene Bcr-Abl

Thank you for submitting your revised manuscript to RSC Chemical Biology. 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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Reviewer 2

The authors carefully addressed all the comments raised by both reviewers. Now, this paper is suitable for publication in RSC Chemical Biology.

Reviewer 1

The authors have considered the reviewers’ comments and have made several changes to the manuscript, including: additional references to mirror-image screening and synthetic D-proteins; increasing the text size in the figures; and toning down the emphasis on mirror-image screening, which will be carried out as a follow-up to this work. I found the text in some of the figures (e.g. the reaction conditions in Fig. 2) to still be rather small. Perhaps a larger figure and different arrangement would allow this text to be bigger in the final formatting. Nevertheless, the changes and responses to the reviewers’ comments have improved the manuscript and I would recommend acceptance as a communication in RSC Chemical Biology.