Chimeric Approach to Identify Molecular Determinants of Nicotinic Acetylcholine Receptors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors' article is written very professionally: it features a solid logical structure and a professional description of nicotinic receptors, specifically the chimeric approach. However, before accepting the paper, the following suggestions from me as a reviewer should be considered, as they are intended to improve the comprehension of the material:

1. The authors understand that the review essentially discusses 10 chimeric examples according to Table No. 1. In my opinion, this is insufficient for a review article. I understand there may not be more chimeras available, but when dealing with a small sample size, it is recommended to discuss it thoroughly and find correlations. Here are the questions that arose for me:
   1.1. Is it possible to draw conclusions from the original publications on how exactly the acquisition of crystalline (or solution) structures of the described chimeras was achieved? That is, not only positive results but also negative experiences. Perhaps the original publications involved several attempts; then describing and discussing failed experiments could help understand how to work better with these receptors.
   1.2. If the described chimeric examples have identical or similar affinity amino acid sequences, it would be interesting to compare how much their obtained quaternary structures differ.
   1.3. It would be helpful to understand from the text immediately which method was used to characterize the structures. Is there a general pattern? Which method is best suited for which case?
   1.4. It would be interesting to compare the experimental conditions and identify the most optimal and simple algorithm for obtaining nicotinic receptor chimeras based on the available literature data.
   The list of questions could be continued, but I think the authors understand my desire—not only to describe but to thoroughly analyze the obtained 10 examples.

2. In section 1.4., it could be indicated to what extent new structural changes in the chimera affect the properties of the original protein. That is, provide examples where creating a chimera significantly affected the original properties and where it did not. Are there any general patterns? It is acceptable to simply refer to existing reviews; I understand this is not the main goal of this work.

3. The reference list does not contain many recent publications. I don't think it's necessary to specifically search for references, but it is worth keeping in mind for future work.

4. Regarding the question of modeling with AI, for example, AlphaFold. It could be discussed in the text how well AlphaFold models such complex systems as receptors. I extend this idea to the question: is creating chimeras still relevant today? Perhaps it is a relevant direction because AI is not yet very good at handling this. But maybe not.

I understand that my questions might significantly extend the work on the publication, and I am not asking the authors to necessarily make changes for each point. However, if the authors wish to provide answers to some of my questions, I believe the publication will become more interesting.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript aims to review the application of chimeric receptor strategies in the study of nicotinic acetylcholine receptors (nAChRs). While the topic is relevant and the manuscript is factually comprehensive, and informative, the review suffers from major conceptual and organizational deficiencies that significantly impair readability, coherence, and scholarly impact. In its current form, the manuscript reads more like a collection of loosely connected background sections rather than a cohesive, insight-driven review. Without substantial restructuring, the manuscript is unlikely to meet the standards expected of a high-quality review article.

 

 

Major Concerns

 

1. Lack of a clear narrative backbone and conceptual hierarchy. A central weakness of this review is the absence of a clearly defined narrative structure. This is particularly evident in section 1.1: Section 1.1 is confusing and lacks a clear storyline from the beginning. The authors initially introduce the chimeric receptor approach in the opening paragraph; however, the discussion then abruptly shifts to general challenges in protein crystallization and structural biology without a clear conceptual or logical connection to chimeric receptors. This transition disrupts the narrative flow and makes it unclear whether the section is intended to motivate the chimera strategy, review structural biology techniques, or introduce receptor-specific challenges. I strongly recommend that the authors substantially rewrite this section to establish a clear and consistent storyline, explicitly linking structural challenges of native receptors to the need for chimeric receptor approaches. Alternatively, if such a connection cannot be clearly articulated, this section (or parts of it) should be removed or significantly condensed. another example is in Section 1.3, the discussion moves from AChBP as a soluble homolog of nAChRs directly to other Cys loop receptors and ELIC receptors. This abrupt shift creates the impression of topic jumping rather than a logically progressing argument. Apperatly, ELIC is most less to employ, we suggest author start with ELIC, then Cys loop, then AChBP.

 

2. Abrupt sentence-level transitions and missing logical connectors. Many paragraphs introduce new concepts without transitional phrases or causal linkage, making the text feel disjointed even when individual sentences are grammatically correct. Like: The sentence beginning “Members of the Cys-loop receptor family, such as gamma-aminobutyric acid…” appears immediately after an extensive discussion of AChBP-based chimeras, yet no transition is provided to explain why the scope is suddenly broadened, which makes people confused about what's the relationship between AChBP and Cys-loop receptor. This pattern occurs repeatedly throughout Sections 1.3-1.5 and the Discussion. The issue is not stylistic but conceptual: the rationale for introducing new receptor systems is often implicit rather than stated, placing an unnecessary cognitive burden on the reader.

 

3. Given the rapid impact of computational structure prediction on receptor biology, I was particularly interested in how recent advances such as AlphaFold have reshaped this field. The authors are encouraged to address whether recent studies have benchmarked or validated AlphaFold-predicted structures against experimentally determined chimera or receptor structures, and how such computational approaches may complement, or potentially reduce the need for—certain chimeric strategies. A more critical and up-to-date discussion of this topic would significantly strengthen the review.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Unfortunately, the version with strikethrough text is very difficult to read. According to MDPI rules, only the new text or edits should have been highlighted. It is not necessary to retain the strikethrough text from track changes mode. The sentence:
"The extent to which structural modifications"
does not start on line 349 as you indicated in the responses to reviewers, but on line 317.
And the same applies to all other edits. Everything has to be searched for manually.

Please check this:
1.3.1. Bacterial homologs:
1.3.2. Cys-loop receptors:
1.3.3. AChBP, an ideal soluble homolog:
I think the ":" symbol is not necessary.

The review is poorly structured in terms of headings. The "Discussion" section is usually omitted in a review article (I mean it can be renamed now, not deleted). In fact, the entire review text is placed within the "Introduction" section. This cannot be the case. It must be corrected. Accordingly, the heading styles and levels need to be changed. I would keep 1.1 as the Introduction, then make 1.2 into 2., 1.3 into 3., and so on.

I ask the authors to correct the headings.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been improved dramatically. I recommend to accept.

Author Response

Thank you very much for your recommendation to accept the manuscript. We greatly appreciate the time and effort you invested in reviewing our work. Your constructive comments have significantly improved the overall quality of this manuscript; it looks much better now.