1. Consolidated peer review report (26 August 2021)

    GENERAL ASSESSMENT

    This preprint reports very high-quality structures of the important transporter, ATP13A2. This work will be of interest to those studying membrane transport, especially scientists studying the P-type ATPases but also those in the channel field working on inward rectifier channels where polyamines give rise to inward rectification due to pore block. There seem to be no structures of any channel solved with polyamine bound.

    The highlight of the manuscript is the structure with a putative substrate bound in the E2-Pi state. After the current preprint was uploaded, a paper by Li et al was published showing several structures of a yeast homolog, Ypk9 (DOI: 10.1038/s41467-021-24148-y). The pose and coordinating residues are similar to those described by Li et al. It is very interesting to see that this ATPase exploits an approach to recognize and bind polyamines similar to that of bacterial periplasmic polyamine-binding proteins, which have different folds and cellular locations (DOI: 10.1002/pro.5560051004;).

    The structures of ATP13A2 in E1 states allow additional understanding of the mechanism and provide some support for a novel mechanism of cytoplasmic polyamine release in which the substrate is released into the cytoplasmic leaflet of the lysosome membrane rather than directly to the cytosol. The Li et al. paper contained only structures in the E2 state, so those authors postulated a half-channel in the E1 state, which this work shows is not there.

    This work was commended by the reviewers for the demonstration, by mutagenesis, that aspartate residues in the binding site are required for productive substrate binding and for characterizing the roles of the N- and C-termini by deletion mutagenesis. In particular, the N- and C-termini of some other P-type ATPases act to inhibit ATPase activity, whereas in ATP13A2, ATP hydrolysis is blocked by removal of the C-terminus but unaffected by removal of the N-terminus.

    For these reasons, we consider this work to be highly meritorious and we anticipate that it will make an important contribution to the P5B-ATPase field.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. The most interesting and novel finding in the manuscript is the evidence that the polyamine substrate may be squeezed out of its binding site into the membrane rather than being released directly to the cytoplasm. According to the conventional thinking about transport by P-type ATPases, the E1 state should have a half-channel from the cytoplasmic surface to the occupied substrate binding site, but the E1 apo structure revealed here has no channel or substrate pocket visible. One explanation might be that there was a transient substrate-bound half-channel which collapsed after substrate dissociation, similar to the one proposed by Li et al. However, the lack of bound substrate in the E1 apo structure argues against this interpretation and also raises the possibility that the substrate is released into the cytoplasmic leaflet of the bilayer. The energetics for such a mechanism appear quite daunting, unless the E2-E1 conformational change disrupts the favorable interactions between bound substrate and the protein, pushing the substrate out of the pocket. Along these lines, does this conformational change cause rotation of the binding site helices in a way that would disrupt favorable interactions between the substrate amino groups and the aspartate residues in the pocket?

      Our recommendation is that the manuscript should not take a strong position in favor of either potential mechanism of release without additional evidence, but rather to present both mechanisms as possibilities.

    2. The role of the terminal domains in ATP13A2 appears to be unique among P-type ATPases. In several other members of this family, these domains serve an auto-inhibitory function by restricting domain movements. However, neither of the ATP13A2 terminal domains acts this way. The N-terminus associates with the N-domain and the membrane, but its deletion has no effect on substrate-dependent ATPase activity. However, the Li et al paper suggested that the N-terminus was auto-inhibitory in Ypk9 and this might be worthy of some comment. The C-terminal extension associates with the P-domain and is apparently required for substrate-dependent ATPase activity either because it's required for ATPase activity or because its removal prevents the transport process coupled to it. In neither of the truncation mutants does ATPase increase by relieving inhibition. Our recommendation would be to discuss the requirements for N- and C-termini in contrast with other ATPases in which the terminal domains are auto-inhibitory (Ann. Rev. Biophys. (2011) 40: 243-266).

    3. The structures revealed a ring of positively charged residues around the interface between protein and lipid on the cytoplasmic surface. This led to the proposal that the region surrounding the putative exit pathway of substrate into the bilayer might be enriched in negatively charged lipids that would help accommodate the released substrate. This feature was not commented on in the paper on the Ypk9 structure. Is it possibly just a fortuitous difference unrelated to mechanism? We recommend that the authors comment on sequence differences between human ATP13A2 and yeast Ypk9 in that region. Mutagenesis experiments could be performed to decrease positive charge in the region adjacent to the putative exit pathway for substrate into the cytoplasmic leaflet. However, the reviewers realize that this might involve much more work than appropriate for inclusion with this manuscript.

    4. The discussion compares P5A and P5B ATPases with the assumption that the substrates for P5A ATPases are mis-targeted N- or C-termini of terminally anchored membrane proteins. Although there is evidence consistent with this function, it has not been demonstrated that P5A ATPase activity is dependent on those mistargeted domains, which still leaves the possibility open that the effect is indirect. The reviewers recommend rewriting this part of the discussion in a way that does not imply that mistargeted terminal domains have been proven to be substrates for P5A ATPases.

    5. As a matter of terminology, occluded states of P-type ATPases, like other transporters, refer to conformations in which the substrate is bound but unable to dissociate because the binding site is closed off to either side of the membrane. This manuscript refers to a substrate-free E1 structure as "occluded". It would be better described as an "empty" or "apo" state.

    Additional suggestions for the authors to consider:

    None

    REVIEWING TEAM

    Reviewed by:

    Wei Mi, Assistant Professor of Pharmacology, Yale University, USA: structural biology, ABC transporter mechanisms

    Michael Broberg Palmgren, Professor, University of Copenhagen, Denmark: mechanism of P-type ATPases

    Gary Rudnick, Professor of Pharmacology, Yale University, USA: mechanism of ion-coupled transporters, biochemical determination of conformational changes

    Curated by:

    Gary Rudnick, Professor of Pharmacology, Yale 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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