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  1. eLife assessment

    This manuscript provides fundamental new insight into protein conformational transitions underlying the transport mechanism of Nramps, an important and widespread transporter family that facilitates the uptake and movement of essential transition metals. Eight new crystallographic structures of the prokaryotic homolog draNRMP in a variety of ligand-bound and conformational states, along with companion molecular dynamics simulations and metal binding and transport assays, provide compelling evidence supporting most of the conclusions. These findings will be of broad interest to scientists studying transport mechanisms and ligand recognition.

  2. Reviewer #1 (Public Review):

    This is a nicely written, very compelling manuscript, comprehensive in scope, that reaches new molecular and mechanistic conclusions on metal transport by Nramp on the basis of extensive crystallographic, molecular dynamics, and metal binding/transport assays. The higher resolution of the structures reported here provides new insights into metal (both Mn and Cd) coordination chemistry along the transport pathway which was generally missing (or incomplete) from previous structural analysis of this well-studied model bacterial system. The findings are strongly topical and likely applicable to other Nramps that are present in higher eukaryotes.

    The new crystallography coupled with the molecular dynamics provides support for the overall transport pathway model. The conclusions are by and large strongly supported by the data. The figures are absolutely outstanding, and readily accessible even to the non-specialist. The authors identify a lower affinity "external" site which may function as an Mn transfer site that kinetically enhances Mn-binding to the cognate "orthosteric" site essential for transport across the membrane.

    Minor weaknesses are the ITC experiments in general. The authors use these experiments to estimate binding affinities of the external and orthosteric sites in a variety of conformations. Although these data are extensive (there are many titrations here), the robustness of the fits to these data is not apparent from what is provided. Clearly the stoichiometry, and thus the binding model (one site vs. two independent sites) was assumed prior to the data fitting; the uncertainties in K are then quite large.

  3. Reviewer #2 (Public Review):

    This study combines data from different experiments to provide a detailed and conclusive mechanism of how transition metal ions are transported by a prokaryotic member of the SLC11 family. Although insight into this process was already provided in previous investigations, the novelty here concerns the presentation of X-ray structures at high resolution which, in combination with previously determined structures of the same protein, show three relevant conformations on the transport cycle in the presence and absence of substrate. For the interpretation of mechanisms, the conclusions derived from these structures are supported by complementary functional experiments from isothermal titration calorimetry and transport assays. Finally, a series of molecular dynamics simulations illustrate the stability of the investigated conformations and the interaction network that was proposed to be relevant for conformational transitions.

    The strength of the manuscript lies in the thoughtful experimental design of the study and the high quality of the data. The X-ray structures are as good as they probably can get for a delicate membrane protein and the interaction with ions was confirmed by anomalous scattering experiments. Although the structure of the outward-facing conformation has relied on a mutation that stabilizes this state, the conformation is similar to known outward-facing conformations of other family members. The presented complementary ITC experiments are of high quality and the experimental design is intriguing.

    A comparably smaller weakness concerns a shortage in the critical assessment of the data and their relation to previous findings in the field. This is in no way meant to question major conclusions drawn from this study, but it might help the reader to better understand the limits of the results and their interpretation. This weakness can be addressed by better documentation of the data and some revision of the text.

  4. Reviewer #3 (Public Review):

    The manuscript by Ray et al. reports a massive body of work targeting the transport cycle of a class of LeuT-fold transporters that specializes in metal transport, the Nramps. The Gaudet laboratory has published extensively on this family of proteins and here they ask the question of how Nramps can transport one of their physiological substrates Mn2+ and how that differs structurally from a toxic metal like Cd2+. The authors capitalize on previously published mutations to trap the transporter in three states with and without Mn2+. Together with ITC data and MD simulations, they put together a plausible, albeit oversold, model of transport. I am not an expert on the details of the technical elements but overall given they appear sound and the corresponding author is a noted expert in crystallography. The structures recapitulate previously seen conformational changes. Nevertheless, the mechanistic story is new and of interest.