PCOMPBIOL-D-24-01553A universal residue numbering scheme for the Immunoglobulin-fold (Ig-fold) to study Ig-Proteomes and Ig-InteractomesPLOS Computational Biology Dear Dr. Youkharibache, Thank you for submitting your manuscript to PLOS Computational Biology. After careful consideration, we feel that it has merit but does not fully meet PLOS Computational Biology's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Comments to the Authors:

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Reviewer #1: The authors provide an updated numbering scheme to identify Ig structural proteomes and interactomes, which allow researchers to communicate about protein structures and function in a standardized fashion. The numbering scheme is innovative in that it does not solely rely on sequence alignments but incorporates the structure and folding patterns. This new scheme is human readable as well as machine readable, allowing for new machine learning algorithms to be implemented.

Comments:

1. The Introduction is a nice review on Ig folds but doesn’t mention the previous numbering schemes until the Results and Discussion section. The information becomes a bit redundant. I would suggest moving some of the discussion in the “Results and Discussion” to the Introduction section to differentiate between older numbering schemes and the contribution of this current work. The “Definition of the IgStrand numbering” section on page 15 seems to be the start of the Results section.

2. Terms are defined later in the article after they are used instead of the other way around. An example is the description of the A strand split on page 12, when it is mentioned on page 6.

3. Page 4, last paragraph, the second sentence is a long incomplete sentence with several references making it difficult to read.

4. Page 15, the scheme is described as using 4 digits, but the 3rd and 4th digit are not discussed until a couple of sections later. A brief mention of all four in the beginning would help with the transitions.

5. Page 6, last paragraph, first sentence: “The immunoglobulin fold is both one and many” is a bit confusing.

6. Figure 3 legend. What does the # represent in the figure? CD4, ICOS, PD-L2, and VNAR domains are mentioned in the legend but not shown in the figure. This should remain in the main text of the article.

7. Table 1. The font should be bigger. It is difficult to read.

8. Page 30, last paragraph, first sentence: The word “least” is written twice.

Reviewer #2: In this work, authors have developed a universal numbering system IgStrand numbering that enables positional comparison of the Immunoglobulin fold (Ig-fold). Ig-fold belong to the Ig Superfamily and is the most populous domain in human genome accounting for 2% of the coding genes. Ig domains are ubiquitous, playing important roles in cellular communications and Immunoglobulin variable domains are the most studied Ig domains.

Ig domains are made of 7-9 strands that make a sandwich of two β sheets. IgStrand numbering (ij50) consists of 4 digits and assigns number 50 to the “anchor residue” in each of the 9 main canonical Ig domain strands: A, B, C, C’, C’’, D, E, F and G. Anchor residues are assigned based on both structural considerations and sequence conservation. Digit “i” spans from 1-9 and represents the canonical strands with their respective order. Digit “j” is usually 5, but strand insertions and splits can be assigned with j+1 or j-1. Residues are number linearly ij50-n or ij50+n if they come before or after the anchor residue.

Using their IgStrand numbering, authors present diverse set of Ig-folds such as Arrestin, Lamin, Transcription factors and Ig-domains in viruses shining light on their structural diversity and variations. Then authors explore Ig-Ig domain interfaces in library of Fab fragments that target SARS-CoV-2 spike protein identifying 361 + 213 unique interacting residue pairs for VH|VL and CH1|CL respectively. Finally, authors explore more complex Ig-chain interfaces such as Tandem IgV1-IgV2 and Ig-Horseshoe domains.

Comments.

• Authors explore an interesting topic, and they managed to come up with a sequence numbering system for the entire Ig Superfamily.

• Authors were able to present their IgStrand numbering in a clear and easy to digest way with nice visual presentation of anchor residues (Figure 6).

• IgStrand numbering is linear and easy to read for humans making comparison of Ig-folds with numerous topostructural variations easier. Furthermore, machine readability of IgStrand numbering could enable comparison of large dataset of Ig domains and quaternary interfaces.

Critical comments:

• Figure 3 a. b. c. is lowercase, but other figures are capitalized such as Figure 5 A, B, C.

• I liked the fact that Authors made IgStrand numbering linear by reserving j for strand insertions and splits (Figure 18 B). A-, and C- are numbered j = 5-1 and A+ and G+ strands are numbered j = 5+1. Although it was mentioned in Page 16 that A’ is a special case and its anchor was numbered j = 5+3 (1850). However, I did not understand why A strand skips j = 7. Was it reserved for something? I think authors should clarify that.

• In Page 30, Authors compare library of 258 Fab fragments. They found 361 unique interacting pairs for VH|VL and they found 231unique interacting pairs for CH1|CL. Since all of these antibodies target the same protein, it is expected to see common interacting pairs for VH|VL even if they might be targeting different epitopes within the same protein. However, considering CH and CL domains are constant, one could expect more common pairs for CH1|CL relative to VH|VL. Authors should further explore this by comparing VH|VL and CH1|CL in library of random Fabs targeting different antigens (ideally, similar size library) to see if common antigen target increases the common residue pairs in VH|VL.

Overall, Authors present interesting and impactful study that can help other researchers in the field. They give enough background to highlight the novelty in their work and their explanation is easy to digest. However, I believe the experiment suggested above is critical to understand the result of IgVH-IgVL and IgCH1-IgCL interactome experiment. Therefore, I believe Authors should address this question along with other two minor comments before publication.

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Review, comments for the Authors.docx

Dear Dr. Youkharibache,

We are pleased to inform you that your manuscript 'IgStrand: A universal residue numbering scheme for the Immunoglobulin-fold (Ig-fold) to study Ig-Proteomes and Ig-Interactomes' has been provisionally accepted for publication in PLOS Computational Biology.

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Iddo Friedberg, Ph.D.

Academic Editor

PLOS Computational Biology

Arne Elofsson

Section Editor

PLOS Computational Biology

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