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  1. Authors' response (4 May 2022)

    GENERAL ASSESSMENT

    The manuscript titled "Structural basis of ion–substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY" by Sauer et al. is an elegant study that details the subtle structural changes of an important secondary-active transporter, VcINDY, upon binding of the co-transported Na+ ions and substrate. The objective of this study was to identify mechanism(s) underlying substrate and ion coupling in the SLC13/DASS transporter family, for which VcINDY serves as prototype and structural model. This is important as several members of the SLC13 family have critical physiological functions, which, when disrupted, can lead to disease in humans.

    This study describes two cryo-EM structures of the Na+-dependent dicarboxylate transporter VcINDY. The key finding is the structure of the inward-apo state (i.e. in the absence of bound Na+ and substrate) of VcINDY, described for the first time. The second structure of the inward-Na+ bound state has an improved resolution of 2.8 Å, allowing better assignment of Na+ at the Na1 and Na2 sites. Together with intrinsic fluorescence measurements, NMR experiments and a cysteine alkylation assay, the study unveils the crucial conformational changes induced by Na+ binding to apo VcINDY and the formation of the substrate-binding site, adding a critical missing piece to the puzzle.

    This is a strong manuscript, which will contribute to our understanding of the SLC13/ DASS family transport mechanism. The structural data are of high quality, and the resolution is in a range that allows precise mechanistic conclusions, down to the identification of a water cluster at the domain interface. The data are presented well, and the manuscript is well-written and easy to read.

    One area for improvement in the manuscript concerns the use of the term "induced fit" to describe what is essentially cooperativity. Our understanding of the manuscript is that "induced fit" is used to describe the mechanism whereby the binding of sodium ion(s) induces the fit of dicarboxylate in the substrate binding site. If this interpretation of the manuscript is correct, it does not align with the traditional meaning of the "induced fit" hypothesis, as put forward by Koshland. In this traditional view, induced fit means that the substrate itself, upon initial binding, changes the structure of its own binding site, to provide better correspondence between the shape and intermolecular interactions of the substrate–binding site complex. In the present manuscript, it is evident that this is the case for Na+ binding (i.e. there is substantial flexibility in the Na+ binding site in the apo state, which decreases upon Na+ binding), whereas the situation is less clear for the dicarboxylate substrate.

    We are grateful to colleagues from Biophysics Colab for reviewing our manuscript. We have revised the manuscript according to their suggestions, along with later revision based on comments from the publishing journal's reviewers. The revised manuscript is stronger and sharper, and we have explicitly thanked the help from Biophysics Colab in the acknowledgement section.

    RECOMMENDATIONS Revisions essential for endorsement:

    We suggest that a more traditional term for describing the formation of the substrate binding site in response to Na+ binding is "cooperativity" of Na+/substrate coupling. There is clearly cooperativity, in the sense that Na+ binding dramatically increases the affinity for substrate, as has been observed for many other Na+-coupled transporters, including those of the SLC1 family, which somewhat share the general elevator transport mechanism with VcINDY. We suggest that the authors rephrase this description, or at least acknowledge the traditional description of "induced fit" mechanism by Koshland (including reference) and clarify the language regarding cooperativity/induced fit models.

    We appreciate the reviewer's highlighting the correct nomenclature to describe our observed sodium-dependent substrate binding. We have revised the manuscript, explicitly stating the "cooperativity" of Na+ and succinate binding. We have also cited a review by Hammes et al (PNAS, 2009) that compares the induced-fit and conformational selection mechanisms.

    In the NMR–style analysis, how many separate refinement runs were performed for the Na+ and Apo structures?

    Each NMR-style analysis used 5 independent runs with NCS turned off, generating 10 independent models. This is been explicitly stated in the revised text, and we appreciate the reviewer's reminder to note this detail.

    Page 7, Results: "VcINDY was found to bind succinate with a Kd of..." This is not a Kd value. The Kd indicates the actual dissociation constant. This value is rather an apparent Km, which is affected by other processes in the transport cycle, in addition to substrate binding.

    We agree with the reviewer that the measured succinate binding may not represent the true Kd for succinate binding, being influenced by other processes in the transporter's conformational cycle. However, the relationship between substrate induced protein changes in vitro versus in vivo is not known. Therefore, we disagree about describing this value as Km, which is the concentration for 50% enzymatic activity rate. We are not measuring directly or indirectly the enzyme turnover (transport rate) in this experiment, and therefore the term Km would be misleading. To better reflect the difference between our in vitro measurement and the biological binding process, we have modified the text to describe it as an "apparent Kd".

    Additional suggestions for the authors to consider:

    In the intrinsic fluorescence assay, insignificant fluorescence change was observed when succinate was titrated to VcINDY without Na+ (Supplementary Fig.2c). To exclude the possibility that VCINDY purified in ChCl does not respond to succinate addition because of loss of protein function during purification, it would be nice to show a positive control. One suggestion is to add NaCl to VcINDY in ChCl, after titration of 1000 μM succinate. A significant fluorescence change comparable to Supplementary Fig.2b would be expected for a positive control. In addition, does the addition of Na+ alone (without succinate) to VcINDY in ChCl cause any fluorescence change? It would be interesting to show.

    The reviewers raise an important caveat regarding the choline binding experiment, and possible control experiment. In ongoing studies, we have performed a similar experiment, measuring Na+ binding for VcINDY purified in choline. Notably, VcINDY's apparent Kd for Na+ is very similar to the previously reported K0.5 in proteoliposomes (Mulligan, JGP, 2014). This indicates VcINDY purified in choline remains properly folded and functional, and the results will be reported in a follow-up study.

    As noted in the manuscript, 3 Na+ ions are co-transported with succinate. In the improved Na+- bound VcINDY structure reported in this study, the Na1 and Na2 sites are well resolved. Asking out of curiosity, do the authors have any thoughts on where Na3 is located on the Na+-bound structure given the data available? In the induced-fit mechanism proposed in the manuscript, are all 3 Na+ ions believed to bind to the Apo transporter before succinate binding?

    The binding site for Na3, and its placement in the substrate binding sequence, is the focus of on-going studies in the lab. However, in previous studies of the homologous human transporter NaDC-1 all Na+ ions were found to bind prior to substrate binding, with the sequence reversed for the release of substrate and sodium. Therefore, the manuscript has been revised to note the unknown role of Na3 in VcINDY's sodium-induced structural changes.

    It is noted that four 3D classes are generated from the VcINDY-choline cryo-EM dataset. The highest resolution class was further refined to 3.2 Å. The resolutions of the remaining three classes are between 3.8 Å and 4.4 Å. May the authors briefly describe what do the other three classes look like? Do they all resemble the same conformation?

    The four, low resolution C1 maps of VcINDY in choline all have distinctly weakened density for HPin and TM10b. However, this varies between maps and protomers, indicating these regions of the protein are in an ensemble of conformations. We have included these maps in the revised manuscript (Supplementary Fig. 7) to illustrate this variation.

    Also, since the transporter is a homodimer and each monomer might function independently, refining the dimer as a whole might not be enough to resolve potential heterogeneous states in the dataset. May the authors discuss if they have tried symmetry expansion and focused 3D classification on one protomer to look for different states, especially in the Apo dataset which may have high heterogeneity? In addition, 3D variability analysis might also be helpful to show the flexibility of the Apo structure.

    The reviewers pose an interesting possibility of processing the VcINDY-choline dataset using more sophisticated techniques to resolve the heterogeneous structures within the Ci-apo state. While we refined the model initially with C1 symmetry to begin to address this prospect, as discussed, we are hesitant to attempt much further. The mobile region in VcINDY is much smaller than targets that have previously undergone this sort of analysis. However, we have deposited the particle stacks into EMDB to enable software development and validation on such challenging cases.

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

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  2. Consolidated peer review report (16 February 2022)

    GENERAL ASSESSMENT

    The manuscript titled "Structural basis of ion–substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY" by Sauer et al. is an elegant study that details the subtle structural changes of an important secondary-active transporter, VcINDY, upon binding of the co-transported Na+ ions and substrate. The objective of this study was to identify mechanism(s) underlying substrate and ion coupling in the SLC13/DASS transporter family, for which VcINDY serves as prototype and structural model. This is important as several members of the SLC13 family have critical physiological functions, which, when disrupted, can lead to disease in humans.

    This study describes two cryo-EM structures of the Na+-dependent dicarboxylate transporter VcINDY. The key finding is the structure of the inward-apo state (i.e. in the absence of bound Na+ and substrate) of VcINDY, described for the first time. The second structure of the inward-Na+ bound state has an improved resolution of 2.8 Å, allowing better assignment of Na+ at the Na1 and Na2 sites. Together with intrinsic fluorescence measurements, NMR experiments and a cysteine alkylation assay, the study unveils the crucial conformational changes induced by Na+ binding to apo VcINDY and the formation of the substrate-binding site, adding a critical missing piece to the puzzle.

    This is a strong manuscript, which will contribute to our understanding of the SLC13/ DASS family transport mechanism. The structural data are of high quality, and the resolution is in a range that allows precise mechanistic conclusions, down to the identification of a water cluster at the domain interface. The data are presented well, and the manuscript is well-written and easy to read.

    One area for improvement in the manuscript concerns the use of the term "induced fit" to describe what is essentially cooperativity. Our understanding of the manuscript is that “induced fit” is used to describe the mechanism whereby the binding of sodium ion(s) induces the fit of dicarboxylate in the substrate binding site. If this interpretation of the manuscript is correct, it does not align with the traditional meaning of the "induced fit" hypothesis, as put forward by Koshland. In this traditional view, induced fit means that the substrate itself, upon initial binding, changes the structure of its own binding site, to provide better correspondence between the shape and intermolecular interactions of the substrate–binding site complex. In the present manuscript, it is evident that this is the case for Na+ binding (i.e. there is substantial flexibility in the Na+ binding site in the apo state, which decreases upon Na+ binding), whereas the situation is less clear for the dicarboxylate substrate.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. We suggest that a more traditional term for describing the formation of the substrate binding site in response to Na+ binding is “cooperativity” of Na+/substrate coupling. There is clearly cooperativity, in the sense that Na+ binding dramatically increases the affinity for substrate, as has been observed for many other Na+-coupled transporters, including those of the SLC1 family, which somewhat share the general elevator transport mechanism with VcINDY. We suggest that the authors rephrase this description, or at least acknowledge the traditional description of “induced fit” mechanism by Koshland (including reference) and clarify the language regarding cooperativity/induced fit models.

    2. In the NMR–style analysis, how many separate refinement runs were performed for the Na+ and Apo structures?

    3. Page 7, Results: "VcINDY was found to bind succinate with a Kd of..." This is not a Kd value. The Kd indicates the actual dissociation constant. This value is rather an apparent Km, which is affected by other processes in the transport cycle, in addition to substrate binding.

    Additional suggestions for the authors to consider:

    1. In the intrinsic fluorescence assay, insignificant fluorescence change was observed when succinate was titrated to VcINDY without Na+ (Supplementary Fig.2c). To exclude the possibility that VCINDY purified in ChCl does not respond to succinate addition because of loss of protein function during purification, it would be nice to show a positive control. One suggestion is to add NaCl to VcINDY in ChCl, after titration of 1000 μM succinate. A significant fluorescence change comparable to Supplementary Fig.2b would be expected for a positive control. In addition, does the addition of Na+ alone (without succinate) to VcINDY in ChCl cause any fluorescence change? It would be interesting to show.

    2. As noted in the manuscript, 3 Na+ ions are co-transported with succinate. In the improved Na+-bound VcINDY structure reported in this study, the Na1 and Na2 sites are well resolved. Asking out of curiosity, do the authors have any thoughts on where Na3 is located on the Na+-bound structure given the data available? In the induced-fit mechanism proposed in the manuscript, are all 3 Na+ ions believed to bind to the Apo transporter before succinate binding?

    3. It is noted that four 3D classes are generated from the VcINDY-choline cryo-EM dataset. The highest resolution class was further refined to 3.2 Å. The resolutions of the remaining three classes are between 3.8 Å and 4.4 Å. May the authors briefly describe what do the other three classes look like? Do they all resemble the same conformation? Also, since the transporter is a homodimer and each monomer might function independently, refining the dimer as a whole might not be enough to resolve potential heterogeneous states in the dataset. May the authors discuss if they have tried symmetry expansion and focused 3D classification on one protomer to look for different states, especially in the Apo dataset which may have high heterogeneity? In addition, 3D variability analysis might also be helpful to show the flexibility of the Apo structure.

    REVIEWING TEAM

    Reviewed by:

    Christof Grewer, Professor, Binghamton University USA: transport mechanisms, kinetics, and structure/function of secondary-active transporters

    Renae M. Ryan, Professor, University of Sydney, Australia: structure and function of membrane transporters

    Xiaoyu Wang, Instructor (O. Boudker lab), Cornell University, USA: membrane transporters, structural biology

    Curated by:

    Renae M. Ryan, Professor, University of Sydney, Australia

    (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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