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Summary:

Cebrero et al. present the structure of TacF, a member of the MOP superfamily. Based on their structural model and MD simulations, they identify key residues that are crucial for the recognition of teichoic acid. The identified residues were validated by in vivo complementation assays. Additionally, they propose a common mechanism of transport for MOP superfamily members based on coevolution and sequence conservation analysis.

Strengths:

The presented structure of the 56 kDa TacF appears to be of high quality for a membrane protein of this size. The BRIL fusion protein Anti-BRIL-Fab strategy used to determine the structure via SPA cryo-EM is innovative and well described in the manuscript. The researchers identified key residues involved in the recognition of teichoic acid using MD simulations. These residues were then validated using an in vivo expression system that allows for systematic analysis of TacF mutants. Taken together, they convincingly describe substrate recognition by TacF, which is an important contribution. In general, the data are explained in a logical way, and the different methods used complement each other to derive a model for the recognition of teichoic acid by TacF. Evolutionary comparisons with other members of the MOP superfamily hint at a similar mechanism.

Major comments:

In the title, the authors state that they have elucidated the “mechanistic basis of teichoic acid transport.” As mentioned above, the data presented are of high quality and convincingly describe the recognition of teichoic acid by TacF.

However, the data presented so far do not fully explain the transport. Clarifying the distinction between transport and recognition in the title and abstract would better align the data with the scope of the manuscript and properly manage readers’ expectations.

To fully describe a mechanism of transport, as in teichoic acid binds on the cytosolic side and is translocated across the membrane, additional experimental evidence is in our opinion necessary. Most of the conclusions regarding the actual transport are drawn from the model protein MurJ and evolutionary comparisons.

Furthermore, some of the evolutionary data lack context. In particular the data in figure 2 do not intuitively help to explain the transport mechanism of teichoic acid. Can proteins from the same cluster compared to proteins from different clusters complement each other in your in vivo assay or MD simulations? What are the differences among their substrates? Maybe this section just needs clarification to make it easier to grasp its significance for the transport mechanism.

To further strengthen the robustness of the in vivo growth assay at least some of the mutants should be cross validated with MD simulation to proof they actually influence substrate recognition.

Minor comments:

Can the authors give any comment or explanation why they only observe the inward conformation?

There is one Ramachandran outlier in the statistics, is it functionally relevant?

What others detergents were tested for the purification, in our experience some detergent soluble substrates co-purify in certain detergents preferentially (GDN, LMNG)

In figure 2 could you please mention what structures are displayed: What organism and if it is an experimental structure or a prediction.

Is it possible to simulate the ethanolamine substrate that was mentioned (“since incorporation of phosphoethanolamine into teichoic acids preserves the key elements recognized by TacF in the repeating unit”) to see if recognition strictly relies on Phosphate groups and not on choline?

An aspect that we would be particularly interested in is the membrane environment surrounding TacF. From the figures it seems like the membrane in the MD simulation is distorted surrounding the protein and bent over the whole length scale of the image. Do these observations have functional significance? Does the hydrophobic tail of teichoic acid compete with certain lipids for binding in the hydrophobic groove?

We generally like the in vivo expression system but had a difficult time to understand the difference between the D39V and VL4012 strain without consulting the supplementary materials. This needs to be better explained it in the text/figure. Also, it is not clear to us on what basis the mutants were grouped in c/d/e (figure 4).

Have you checked relative expression levels of the tested mutants to exclude protein instability as a cause of the observed growth defect (figure 4)?

Why was the R230 R333 double mutant not tested in the growth assay (figure 4)?

Is it plausible that close MurJ variants (figure 5 d) complement or in part rescue a TacF k/o growth defect?

In figure 5 the turn of the models is a bit misleading since they are not both turned in the same way (+/- 90°)

The long-range interactions (figure 5) that presumably are important for stabilizing the outward conformation could be tested with the established growth assay.

“The flipping of teichoic acid represents a rate-limiting step” there is no evidence for this statement in the discussion, also no source is given if it is already published data.

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.