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  1. Endorsement statement (5 August 2022)

    Peter et al. describe the first experimentally validated structural model of a canonical member of the TRAP family of transporters, Haemophilus influenzae (Hi)SiaPQM, which transports sialic acid into bacteria. By elegantly combining a cryo-EM structure of the HiSiaQM dimer, AlphaFoldmodels, and sequence, biochemical, and mutational analyses, the authors shed light on the fold and domain organization of the tripartite HiSiaPQM holocomplex. The authors also propose a structure-based model for its transport mechanism: substrate recognition is "outsourced" to the substrate binding protein (P protein) by the QM proteins, which in turn use an elevator mechanism to transport sialic acid across the membrane. The work is rigorous and convincing, and it presents valuable findings that will be of interest to scientists investigating transporters with an elevator-type mechanism as well as membrane transport more generally.

    (This endorsement by Biophysics Colab refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

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  2. Authors' response (4 August 2022)

    GENERAL ASSESSMENT

    Tripartite ATP-independent periplasmic transporters (TRAPs) are found in many bacterial and archaeal organisms. Some TRAPs are essential for survival of pathogenic microorganisms (e.g., H. influenzae and V. cholerae). TRAPs are secondary active transporters that couple the uptake of substrate to the symport of two sodium ions. TRAPs have membrane-embedded Q & M domains, and a soluble substrate-binding protein (SBP or P), which captures and delivers substrates to the QM domains. Whereas the structures of some SBPs were known, no structural information was available for the membrane-embedded QM domains of a TRAP transporter.

    This manuscript by Peter and colleagues describes the 3D architecture of a canonical member of the TRAP family of transporters, Haemophilus influenzae (Hi)SiaPQM, a sialic acid transporter, using a combination of experimental structures and structure predictions. They determined a cryoEM structure of the transmembrane component, HiSiaQM, in lipid nanodiscs at medium resolution (the actual resolution is unclear in the original version of the manuscript). To overcome the challenge that HiSiaQM has a molecular weight of 70 kDa, too small for straight-forward single-particle Cryo-EM imaging, the authors generated single variable domain on heavy chain (VHH) antibodies, which inhibit the transporter in cell-based assays. They then generate a Megabody using one of them and decorated HiSiaQM to facilitate particle picking and alignment. The authors are cautious in their interpretation of the map and use Alphafold to aid model building. HiSiaQM forms a monomeric elevator-type fold in which the "M" module has homology to the VcINDY substrate-binding domain, and the "Q" module forms an extended stator domain. The HiSiaQM structure is in an inward-open conformation, and they use a structure of VcINDY to predict an outward-open conformation. Using AlphaFold, they also generate a prediction for how the SBP or "P" module, the periplasmic sialic acid binding protein, interacts with the QM transmembrane component. They use a complementation assay to validate aspects of their model by determining the functional consequence of mutations at key positions.

    This elegant study combines diverse approaches to develop compelling mechanistic hypotheses. The manuscript provides the first experimentally validated structural model of a full TRAP transporter, shedding light on its fold and domain organization. The new structure and models also allow the authors to propose a more detailed and structure-based working model for its transport mechanism: substrate recognition is "outsourced" to the SBP by QM, which then uses an elevator mechanism to transport sialic acid across the membrane.

    The method section is exemplary in detail, and much of the biochemical data (protein preparations and in vitro VHH or sialic acid binding assays) are of high quality. The assay to probe from which side the VHH binders inhibit the transporter is cleverly designed. The cryoEM structure is of medium resolution, but by using predicted folds and homology to other elevator-type transporters, the authors arrive at a well-supported model, although with the current presentation of the data, it is unclear to what extent the model is accurate in its details. The sequence, biochemical, and mutational analyses are not extensive in scope, although they are useful to support the structural model. With the tools and reagents available to the authors, it is somewhat surprising that they used a Megabody instead of the SBP to increase the size of the particles (e.g., it seems that they could use the same SPR assay they used to measure nanobody binding to search for conditions that promote SBP binding to the QM domain). Therefore, an experimental structure of the full HiSiaPQM transporter awaits future studies.

    Thank you for this overall positive assessment of our manuscript.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. The study's main limitation is the low resolution of the maps and the consequent need to rely on modeling, which likely recapitulates the overall fold well but might fail on details. Therefore, it is important to document how good the model is and how informative the data are in critical protein regions. Specifically, the authors should show close-ups of the model placed into the density for regions such as expected sodium and substrate binding sites. Also, the authors should show the fit of individual TM helices to substantiate the helical register. Submitting the full coordinates to the Protein Data Bank might be inappropriate if there are significant uncertainties about the structure.

    We agree. In the meantime, further processing by a combination of RELION and cryoSPARC has significantly improved the quality of our 3D reconstruction. We can now see conclusive density for many aromatic sidechains that confirm the register of the helices. As suggested by the referees, we provide close-up figures with density in the supporting information (Figure S4, S5).

    We have included these data in a revised version of Fig.1 in the published article:https://www.nature.com/articles/s41467-022-31907-y

    1. A related issue is that the resolution of the cryoEM structure needs to be clarified: it is stated as 6.2 Å in the main text (page 4, line 18) and the methods, but Fig S4c shows the resolution as 6.84 Å, and Table S1 lists it as 4.5 Å. Similarly, the authors should indicate clearly in the main text which map was used for model building: The authors use their low-resolution map for model building (page 4 line 18), but show that they obtain a higher resolution map by subtracting the signal corresponding to lipid nanodisc (Fig. S4 d,e). The authors should also show their subtracted map at higher contour in Fig. S4, it is not possible to judge the quality of the map at present. As mentioned above, a supplementary figure with individual helices fitted into cryo-EM map would be helpful.

    As mentioned in the point above, the data has been reprocessed, now using a combination of cryoSPARC and RELION as shown in Figure S3. We used the particle subtracted map for the model building, which has a GSFC resolution of 4.7 Å. This is now clearly stated in the text. The requested figure with individual helices is shown in the published article:https://www.nature.com/articles/s41467-022-31907-y

    1. Section "Experimental validation of the tripartite model": The rationale for choosing mutations is not sufficiently explained, i.e., what is the importance of the periplasmic loop? What are the expected interactions between SBP and QM? It would make it clearer to explain the "scoop-loop" model and the expected P-QM interactions at the very beginning of the section.

    Thank you for the suggestion. We have now explained the rational for the selection of the mutants directly at the beginning of this section of the manuscript:

    "To validate the described structures, we selected 31 residues in regions that we thought are important for the integrity and function of the TRAP transporter, such as the substrate-binding site of the P-domain (I), the extended periplasmic loops of the Q domain (II), the P-QM interface (III and IV), or the assumed sialic acid- and Na+ binding sites at HP1 and HP2 (V) of the QM domains (Figure 5a) (detailed views of the mutation sites are shown in Figure S14 and a sequence alignment of TRAP transporters in Figure S15 and S16). Sites in the periplasmic loops of the Q domain were selected to test for a potential "scoop-loop" mechanism, as found in SBP-dependent ABC transporters 60. The effects of all mutants were analyzed in the above-described SEVY3-based complementation assay."

    It would also help to add interpretation of some of the phenotypes (i.e., for mutants D58R, S60R, E172R, R30E, S356Y, E429R). Overall, the discussion of the location these mutations seems underdeveloped. Finally, it would be useful to have an additional panel in Figure 3 (or edited versions of panels a and b) that indicate on the structural models the position of the mutations that impair sialic acid transport.

    We have added a discussion of the individual mutants in the main text and have improved Figure 5, so that the structural impact of the mutants can be better judged by the reader. We have included the new Figure in the published article:https://www.nature.com/articles/s41467-022-31907-y

    1. All binding and activity measurements should have an estimation of errors (and a description of what the error bars are) and reproducibility verification. All measurements need replicates and corresponding statements in the figure legend or methods.

    Done.

    1. The authors should carefully review and revise their references as needed. For example, when discussing other elevator-type transporters, the authors should refer the structural papers as those were the papers that established the elevator-type mechanism. Also, the authors should reference the structural study on the outward-facing conformation of DASS transporters (https://elifesciences.org/articles/61350).

    The references have been added.

    1. Figure S10: it is unclear how the data for VHHQM4 are interpreted as all VHHs binding except for VHHQM5.

    This is because VHHQM4 and VHHQM5 bind to the same region of the QM domains and hence mutually exclusive.

    Additional suggestions for the authors to consider:

    1. The uniqueness of HiSiaPQM could be better emphasized. Isn't it the first example of a monomeric elevator-type transporter?

    Indeed. We have now emphasized this a bit more.

    1. It might be beneficial to swap the first two sections of the paper and first describe binders selection and characterization, then the rationale for choosing VHHqm3 for structural work, and the resulting structure.

    We thought about this a lot, but decided to start the manuscript with a description of the TRAP structure.

    1. Since subtraction of the nanodisc signal improved the resolution of the reconstruction, the authors could try masked classification and refinement of the low-resolution map, including trying other software packages for refinement/classification and masking. Generally, several rounds of ab initio/3D classification are often required to obtain a clean particle stack. If the authors did this, they should indicate as such. The authors may also find it useful to adjust the dynamic masking parameters during refinement in cryoSPARC. Membrane proteins seem to not be compatible with cryoSPARC default values and require adjustment. This may result in cleaner, more accurate FSCs.

    Thank you for the suggestion. In the meantime, we have reprocessed the dataset and found that 3D classification in RELION improved the particle stack significantly. We performed one round of 3D classification with alignment, then applied a protein mask and performed another round of 3D classification without alignment. The procedure is described in the updated workflow figure S3 which is also pasted above.

    For the local refinement in cryoSPARC we used static masks, as is the default of version 3.1. Dynamic masking is to our knowledge not recommended for local refinements.

    1. Page 6, the authors title the section "High-affinity VHHs reveal the membrane orientation…": was the membrane orientation a matter of debate? Also, this result is only mentioned in passing in that section. We suggest editing the section title or the section text to make the two more consistent with each other.

    While this point was not a matter of debate, there was nevertheless no experimental evidence for it and we thought it worth mentioning. We have removed the statement from the section heading.

    1. Page 6 line 16. Can the authors describe the criteria used to adjust the loops if there is no experimental density for the outward-facing conformation?

    The geometry of the loops was relaxed using the regularize feature of Coot. This information has been added to the paper.

    "The loops connecting elevator and stator domains were adjusted manually using the geometry regularization feature in Coot 49."

    1. In the section on the model of the tripartite transport complex (or perhaps in the following section, in which they describe their experimental validation), the authors should mention that the interface of the periplasmic domain with the transmembrane module has the lowest certainty (Figure S3).

    Reprocessing of our dataset resulted in a much better-defined density in this region, see above.

    (Related to this, on page 10 line 34, and page 11 line 2, the authors use "perfect" and "perfectly" to describe the fit and match of this interface, which seems overstated considering the evidence available.)

    True. We have toned the statement down.

    Two other statements warrant further discussion: (i) Why do the authors postulate that both lobes of the SBP would remain connected to the QM protein in both the outward-open and inward-open conformation?

    In our working hypothesis, the transporter preferably recognizes the closed state of the SBP. For substrate translocation from the SBP to the transporter, both the transporter and the SBP have to open. The hypothesized conformational coupling of SBP and transporter (as shown in Figure 4) is an elegant way of achieving this. After the substrate translocation the SBP will of course dissociate from the transporter.

    We have included single molecule microscopy data to the revised manuscript, which further supports our mechanism.

    (ii) Can the authors propose any reason why it would be beneficial to have the dimeric SBP oriented as they predict, with one facing away from the membrane?

    We used this example as a way of validating the proposed orientation of the P domain on the transporter. The fact that the second monomer of the SBP dimer does not clash into the transporter supports our model. Our model cannot explain the necessity of a dimeric SBP.

    1. It could be nice to discuss the inhibitory effect of nanobodies using the structural information. Can SBP still dock when the nanobody is bound, as seen in the structure? Perhaps this is a basis for inhibition of the transporter from the periplasmic side, and could be similar for other binders that inhibit HiSiaPQM?

    We now discuss this in the manuscript. Indeed, the inhibitory effect of VHHQM3 can be explained by our structure because it blocks the SBP binding site and interlocks the stator and elevator domains of the transporter.

    1. From Figure 1D and S7, it is difficult to judge how similar are vcINDY and HiSiaPQM in detail around HP1 and HP2. Thus, the conclusion that sodium ions likely bind between the HPs seems unsubstantiated. A focused representation of the structural alignment around the proposed sites and discussion of the relevant residues and their conservation might strengthen the hypothesis.

    This has been done in the new Figure 2 in the published article: https://www.nature.com/articles/s41467-022-31907-y

    (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

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  3. Consolidated peer review report (19 February 2022)

    GENERAL ASSESSMENT

    Tripartite ATP-independent periplasmic transporters (TRAPs) are found in many bacterial and archaeal organisms. Some TRAPs are essential for survival of pathogenic microorganisms (e.g., H. influenzae and V. cholerae). TRAPs are secondary active transporters that couple the uptake of substrate to the symport of two sodium ions. TRAPs have membrane-embedded Q & M domains, and a soluble substrate-binding protein (SBP or P), which captures and delivers substrates to the QM domains. Whereas the structures of some SBPs were known, no structural information was available for the membrane-embedded QM domains of a TRAP transporter.

    This manuscript by Peter and colleagues describes the 3D architecture of a canonical member of the TRAP family of transporters, Haemophilus influenzae (Hi)SiaPQM, a sialic acid transporter, using a combination of experimental structures and structure predictions. They determined a cryoEM structure of the transmembrane component, HiSiaQM, in lipid nanodiscs at medium resolution (the actual resolution is unclear in the original version of the manuscript). To overcome the challenge that HiSiaQM has a molecular weight of 70 kDa, too small for straight-forward single-particle Cryo-EM imaging, the authors generated single variable domain on heavy chain (VHH) antibodies, which inhibit the transporter in cell-based assays. They then generate a Megabody using one of them and decorated HiSiaQM to facilitate particle picking and alignment. The authors are cautious in their interpretation of the map and use Alphafold to aid model building. HiSiaQM forms a monomeric elevator-type fold in which the “M” module has homology to the VcINDY substrate-binding domain, and the “Q” module forms an extended stator domain. The HiSiaQM structure is in an inward-open conformation, and they use a structure of VcINDY to predict an outward-open conformation. Using AlphaFold, they also generate a prediction for how the SBP or “P” module, the periplasmic sialic acid binding protein, interacts with the QM transmembrane component. They use a complementation assay to validate aspects of their model by determining the functional consequence of mutations at key positions.

    This elegant study combines diverse approaches to develop compelling mechanistic hypotheses. The manuscript provides the first experimentally validated structural model of a full TRAP transporter, shedding light on its fold and domain organization. The new structure and models also allow the authors to propose a more detailed and structure-based working model for its transport mechanism: substrate recognition is “outsourced” to the SBP by QM, which then uses an elevator mechanism to transport sialic acid across the membrane.

    The method section is exemplary in detail, and much of the biochemical data (protein preparations and in vitro VHH or sialic acid binding assays) are of high quality. The assay to probe from which side the VHH binders inhibit the transporter is cleverly designed. The cryoEM structure is of medium resolution, but by using predicted folds and homology to other elevator-type transporters, the authors arrive at a well-supported model, although with the current presentation of the data, it is unclear to what extent the model is accurate in its details. The sequence, biochemical, and mutational analyses are not extensive in scope, although they are useful to support the structural model. With the tools and reagents available to the authors, it is somewhat surprising that they used a Megabody instead of the SBP to increase the size of the particles (e.g., it seems that they could use the same SPR assay they used to measure nanobody binding to search for conditions that promote SBP binding to the QM domain). Therefore, an experimental structure of the full HiSiaPQM transporter awaits future studies.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. The study's main limitation is the low resolution of the maps and the consequent need to rely on modeling, which likely recapitulates the overall fold well but might fail on details. Therefore, it is important to document how good the model is and how informative the data are in critical protein regions. Specifically, the authors should show close-ups of the model placed into the density for regions such as expected sodium and substrate binding sites. Also, the authors should show the fit of individual TM helices to substantiate the helical register. Submitting the full coordinates to the Protein Data Bank might be inappropriate if there are significant uncertainties about the structure.

    2. A related issue is that the resolution of the cryoEM structure needs to be clarified: it is stated as 6.2 Å in the main text (page 4, line 18) and the methods, but Fig S4c shows the resolution as 6.84 Å, and Table S1 lists it as 4.5 Å. Similarly, the authors should indicate clearly in the main text which map was used for model building: The authors use their low-resolution map for model building (page 4 line 18), but show that they obtain a higher resolution map by subtracting the signal corresponding to lipid nanodisc (Fig. S4 d,e). The authors should also show their subtracted map at higher contour in Fig. S4, it is not possible to judge the quality of the map at present. As mentioned above, a supplementary figure with individual helices fitted into cryo-EM map would be helpful.

    3. Section “Experimental validation of the tripartite model”: The rationale for choosing mutations is not sufficiently explained, i.e., what is the importance of the periplasmic loop? What are the expected interactions between SBP and QM? It would make it clearer to explain the “scoop-loop” model and the expected P-QM interactions at the very beginning of the section. It would also help to add interpretation of some of the phenotypes (i.e., for mutants D58R, S60R, E172R, R30E, S356Y, E429R). Overall, the discussion of the location these mutations seems underdeveloped. Finally, it would be useful to have an additional panel in Figure 3 (or edited versions of panels a and b) that indicate on the structural models the position of the mutations that impair sialic acid transport.

    4. All binding and activity measurements should have an estimation of errors (and a description of what the error bars are) and reproducibility verification. All measurements need replicates and corresponding statements in the figure legend or methods.

    5. The authors should carefully review and revise their references as needed. For example, when discussing other elevator-type transporters, the authors should refer the structural papers as those were the papers that established the elevator-type mechanism. Also, the authors should reference the structural study on the outward-facing conformation of DASS transporters (https://elifesciences.org/articles/61350).

    6. Figure S10: it is unclear how the data for VHHQM4 are interpreted as all VHHs binding except for VHHQM5.

    Additional suggestions for the authors to consider:

    1. The uniqueness of HiSiaPQM could be better emphasized. Isn’t it the first example of a monomeric elevator-type transporter?

    2. It might be beneficial to swap the first two sections of the paper and first describe binders selection and characterization, then the rationale for choosing VHHqm3 for structural work, and the resulting structure.

    3. Since subtraction of the nanodisc signal improved the resolution of the reconstruction, the authors could try masked classification and refinement of the low-resolution map, including trying other software packages for refinement/classification and masking. Generally, several rounds of ab initio/3D classification are often required to obtain a clean particle stack. If the authors did this, they should indicate as such. The authors may also find it useful to adjust the dynamic masking parameters during refinement in cryoSPARC. Membrane proteins seem to not be compatible with cryoSPARC default values and require adjustment. This may result in cleaner, more accurate FSCs.

    4. Page 6, the authors title the section “High-affinity VHHs reveal the membrane orientation…”: was the membrane orientation a matter of debate? Also, this result is only mentioned in passing in that section. We suggest editing the section title or the section text to make the two more consistent with each other.

    5. Page 6 line 16. Can the authors describe the criteria used to adjust the loops if there is no experimental density for the outward-facing conformation?

    6. In the section on the model of the tripartite transport complex (or perhaps in the following section, in which they describe their experimental validation), the authors should mention that the interface of the periplasmic domain with the transmembrane module has the lowest certainty (Figure S3). (Related to this, on page 10 line 34, and page 11 line 2, the authors use “perfect” and “perfectly” to describe the fit and match of this interface, which seems overstated considering the evidence available.) Two other statements warrant further discussion: (i) Why do the authors postulate that both lobes of the SBP would remain connected to the QM protein in both the outward-open and inward-open conformation? (ii) Can the authors propose any reason why it would be beneficial to have the dimeric SBP oriented as they predict, with one facing away from the membrane?

    7. It could be nice to discuss the inhibitory effect of nanobodies using the structural information. Can SBP still dock when the nanobody is bound, as seen in the structure? Perhaps this is a basis for inhibition of the transporter from the periplasmic side, and could be similar for other binders that inhibit HiSiaPQM?

    8. From Figure 1D and S7, it is difficult to judge how similar are vcINDY and HiSiaPQM in detail around HP1 and HP2. Thus, the conclusion that sodium ions likely bind between the HPs seems unsubstantiated. A focused representation of the structural alignment around the proposed sites and discussion of the relevant residues and their conservation might strengthen the hypothesis.

    REVIEWING TEAM

    Reviewed by:

    Olga Boudker, Professor and HHMI Investigator, Weill Cornell Medicine, USA: structure and mechanism of membrane transporters

    Rachelle Gaudet, Professor, Harvard University, USA: structure and mechanisms of transporters

    Valeria Kalienkova, Postdoctoral Researcher (C. Paulino lab, University of Groningen, Netherlands): single particle cryo-EM, x-ray crystallography, membrane proteins

    Krishna Reddy, Postdoctoral Researcher (O. Boudker lab, Weill Cornell Medicine, USA): single-particle cryo-EM, membrane protein structure and mechanism

    Curated by:

    Rachelle Gaudet, Professor, Harvard University, USA

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

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