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The manuscript by Cebrero et al. investigates the mechanism by which the Streptococcus pneumoniae teichoic acid flippase, TacF, recognizes the phosphocholine content of teichoic acids to ensure only fully modified subunits are transported. To determine the mechanistic basis of teichoic acid recognition, the authors utilize cryo-electron microscopy to determine the structure of TacF reconstituted in lipid nanodiscs mimicking the S. pneumoniae cell membrane, perform evolutionary coupling analysis, and utilize molecular dynamic simulations coupled with in vivo functional characterization to identify residues important for teichoic acid binding. Finally, the authors contextualize their findings by comparing TacF to other members of the multidrug/ oligosaccharide-lipid/polysaccharide (MOP) superfamily, suggesting other flippases within this family may recognize substrates via a similar mechanism. Overall, we feel that the data presented within this manuscript is well done and largely supports the claims made by the authors. The consistent color-coding throughout the figures increased audience accessibility, and the model summarized the proposed mechanism well. We outline major and minor adjustments aimed at strengthening the data provided and improving clarity for a broader audience.

Major comments

-While we found the data compelling, the main focus of this study appears to be a structural, mutational, and conservation analysis of the TacF protein suggesting a conserved flippase mechanism as opposed to solving the mechanistic basis of the teichoic acid transport. We recommend altering the title of the manuscript to better reflect the data presented. One such alteration would be “Structural, evolutionary, and mutational analysis of TacF suggests a conserved flippase mechanism.” Additionally, we recommend changing the title of Figure 6 to “Proposed mechanistic model of teichoic acid flipping by TacF” because binding and/or transport was not directly experimentally tested in this study.

-The titles of each figure and section were not summarizing the conclusions drawn from the data presented. We recommend rewriting the titles of each section and figure to improve paper flow and guide readers naturally through the text.

-The schematic provided in Figure 4B is excellent and greatly improves the clarity of the figure. We suggest including all residues which are mutated in 4C-E in the schematic provided in 4B. For example, R152, F227, and Y38 are mutated but not shown in 4B.

- While the data shown in Figure 4C-D, indicating certain double and triple alanine substitutions of residues identified in the molecular dynamics simulations shown in Figure 3 is convincing, this data could be strengthened by confirming that the proteins harboring these point mutations are stable. The authors pose the hypothesis that only double and triple substitutions impacted growth due to cooperation of multiple residues needed to recognize the large teichoic acid precursors. It is also possible that double and triple substitutions of alanine for charged and/or aromatic residues destabilize the protein, which would give the same growth readout. This could be directly addressed in the text.

- Certain combinatorial point mutants could further strengthen the finding that disrupting the binding of phosphate groups of phosphocholine and GalNac units compromise cell growth (lines 250-252). For example, including a protein harboring a double R230A and R333A mutation, rather than with the R250A mutation as is currently shown in Figure 4C, would inform solely on the phosphocholine interaction. Another example would be including mutations in R15 and R269 alone and combinatorially to inform on the diphosphate moiety interaction, particularly because these residues are discussed extensively throughout the manuscript. The study could incorporate the growth phenotype of the aforementioned mutants to adequately support the conclusion that these residues are involved in recognition of the teichoic acid, or the text could be revised to ensure the conclusions tightly align to the findings currently included.

- We were surprised to see growth of the VL4012 strain when no inducer was present (Figure 4A, yellow line), along with the growth of the strains in Supplementary Figure 7. It had been previously stated that deletion of TacF was lethal in S. pneumoniae. Is the growth observed due to suppressor mutations, such as in the Plac promoter which would allow constitutive TacF expression? Or do TacF mutants often recover following an initial growth defect? The growth defect from hours 0-8 is striking, but the growth following this period could be addressed when first introducing the inducible system shown in 4A.

-Figure 3 specifies that the role of residues R15 and R269 is to coordinate the diphosphate linker during recognition of the teichoic acid. However, Figure 3C shows that residue 15 is equally likely to be an asparagine or an arginine in the TacF homologs. This variation seems like it would affect the ability of TacF to properly align and recognize the molecule since the two amino acids vary in their physical properties (R is positively charged and larger, N is amidic and smaller). It is also unclear where the sequences portrayed in the sequence logo analysis originated from; are these sequences including proteins that are not true TacF homologs? Please include a more thorough description of how sequence logos were generated in the figure legend, and address the R and N dichotomy occurring at the R15 residue and how it may potentially impact the ability of TacF to recognize the teichoic acid.

Minor comments

-In the text, figures are referred to with capital letters while on the figures themselves the sections are denoted with lowercase letters.

- The LicB protein depicted within the model shown in Figure 1A is not mentioned in the text. A brief description of the protein’s function is needed to contextualize it with the other proteins shown. Alternatively, the LicB protein could be removed from the model and replaced with example choline-binding proteins (CBPs) which are specifically mentioned in the introduction and discussion sections.

-Figure 1A, line 858: Define the P-C in the diagram as being the phosphocholine modifications.

-Figure 1F-H: Including an arrow to show how the orientation of the protein has changed between panels (as done in Figure 5B/C) would increase clarity. Additionally, the boxes shown in Figure 1H do not align exactly with the boxes shown in the cytoplasmic view of the protein shown in Figure 1F directly above 1H. The cartoon model of the 14 transmembrane domains in 1F is very helpful in understanding how the protein is oriented in the cellular membrane, but the cartoon is flipped when compared with the protein structure directly below it.

-The representative homologues used in Figure 2 should be explicitly named and PDB identifiers provided within the figure.

-Figure 4A: As written in the figure caption on line 920, it is not immediately clear that D39V is a true WT strain, while VL4012 is the WT strain with the double expression system. Please consider more directly stating this in the figure caption.

-Figure 4C-E: The x-axis label font size could be increased to improve legibility. The figure legend should also state how graphs C, D, and E differ from each other. Should nine datapoints be shown for the three technical replicates per three biological replicates? Some samples appear to have fewer data points than others.

-Supplemental Figure 5D, which depicts and numbers the specific amino acids that are referenced over the course of the manuscript, was incredibly helpful for understanding where these amino acids are relative to each other and where they are relative to the distal and proximal site and the groove domain. Adding this panel to Figure 4 would serve as a good reference for readers unfamiliar with this protein to quickly reference which amino acids are being discussed and where these amino acids are on the protein.

-Figure 5A: Should red letters be GalNac rather than GlcNac? It appears this nomenclature may have been switched elsewhere in the manuscript as well (Line 246, 251, 258, etc.).

-Figure 5B-C: At first glance, it is not clear which orientation is the inward –facing and which is the outward-facing conformation. Adding lines with labels indicating the two sides of the membrane similar to Figure 1F would help improve reader comprehension.

-Lines 95-96 describe “BRIL”, a fiducial marker used to aid in structural characterization. It would be helpful to further explain why this specific marker was chosen, both to contextualize how these markers function and to highlight the fact that such markers are widely used in this type of work.

-Lines 101-121 detail the process of determining which construct to proceed with for further analysis using cryo-EM, but the process was difficult to follow. It would improve the flow of this passage to explain which tests were included and why before explaining the results of each screen, as listing them out in a chronological order makes it confusing as to why models that seem to be incompatible with this analysis were not ruled out and were instead included in subsequent screens.

-Lines 125-126 could state the native ratio of phosphatidylglycerol to glycolipids in the S. pneumoniae membrane (if known) and whether the nanodiscs were representative of this ratio.

-Line 232 states “the first one...” when describing the two constructs introduced into the tacF knock-out strain. Replacing “...while the other...” on line 233 with “...the second one” would improve reader comprehension and emphasize the identity of the two constructs.

-Lines 261-262 contain a sentence fragment; changing the period to a comma would fix this.

Carter Collins and Camy Guenther (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Clay Fuqua, Josy Joseph, Lily Pumphrey and Tahreem Zaheer. We are part of the Dept. of Biology where Malcolm E. Winkler’s group is located. Malcolm is a coauthor on a recent publication with one of the corresponding authors (Jan-Willem Veening); this prior interaction did not influence the choice of this preprint for our class.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.