Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9

Abstract Na+/H+ exchangers (NHEs) are ancient membrane‐bound nanomachines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharmaceutical drugs. Despite their fundamental importance to cell homeostasis and human physiology, structural information for the mammalian NHE was lacking. Here, we report the cryogenic electron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and attention deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion‐binding site are remarkably similar to distantly related bacterial Na+/H+ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion‐binding site, their elevator‐like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications.

Please remember: Digital image enhancement is acceptable practice, as long as it accurately represents the original data and conforms to community standards. If a figure has been subjected to significant electronic manipulation, this must be noted in the figure legend or in the 'Materials and Methods' section. The editors reserve the right to request original versions of figures and the original images that were used to assemble the figure.
Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide The revision must be submitted online within 90 days; please click on the link below to submit the revision online before 3rd Nov 2020. Winkelman et al. & Drew working in collaborat ion wit h the Beckst ein and Robinson groups have characterised horse NHE9 though a combination of methods -yeast expression, cryo-EM structure, functional assays in proteoliposomes using proton-sensitive fluorescence, biophysics of GFPprotein stability, CG-MD and homology models. The level of conclusions on ion binding sites and the putative mechanism are justified from literature and the experiments presented, and they discuss the relevance to understanding for example autism-spectrum disorders. This is fine, solid work, basically impeccable, and it shoudl interest a large readership. I have only few remarks: 1) the introduction. "Dysfunction of NHEs has been linked to diseases such as ....." -perhaps be a bit more specific on how various diseases are either caused by NHE dysfunction, or in other cases correlated with NHE upregulation/downregulation 2) the introduction: "Human disease mutations of NHE9 are linked to neurological disorders such as familial autism, ADHD and epilepsy, making NHE9 a prime drug target8-10". This remark is a bit too easy -how could a drug targeting NHE9 overcome these disorders if they are caused by NHE9 mutations and dysfunction?

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3) The disordered C-terminus could perhaps be addressed a bit more except to say that it is disordered as shown for the isolated NHE1 tail (refs in the paper). Please include it in the sequence alignment (suppl. fig. 1). Could interactions occur under certain functional states that are not imposed here? It would be too much to ask for more structures that address the C-terminal interactions, but it will be highly interesting to discuss putative mechanisms of auto regulation and how they may interfere with the transport models presented here, or even disease mutations.
Referee #2: Proton/sodium ion exchangers (NHEs) are essential membrane transporters required to maintain the intracellular or intraorganellar pH, the sodium ion concentration and the volume. The manuscript describes the structure of a mammalian electroneutral proton/sodium ion exchanger (NHEs), namely NHE9 from horses, as determined by cryoEM. This is a major achievement. Many groups tried to achieve this goal but failed because of lack of protein production or insufficient stability of the produced protein. Key for the success of the authors was a careful comparative screening of 12 NHE9s from various vertebrates, identifying the most appropriate NHE for their studies. Apart from the structure the authors show by using mass spectrometry the importance of phosphoinositides for the dimerization of NHE9 and its activity. They use molecular dynamics simulations and related methods to investigate the mechanism. Because the methodological repertoire is very broad an appropriate evaluation of the manuscript would require experts from these various fields. My major concerns are twofold: -The quality of the structure, as indicated by the pdb validation report is low. In particular there are many clashes of side chains, and non-hydrogen atoms outside of the density. The authors have to improve the structure or provide an explanation for its low quality.
-Residue D244 is considered to be essential for binding the transported sodium ion. The substantial activity of variant N243A-D244A (figure 1C) is highly surprising. This fact is not mentioned nor discussed in the text. In my opinion this observation invalidates a major part of the mechanistic discussion where D244 plays the key role for the exchange activity. I suggest to use MD simulations and binding calculations to investigate whether a sodium ion still can bind near D244A. I cannot judge the quality of the Elastic Network Modelling (ENM) and transition pathway generation. Minor concerns: p.3 and methods: Please explain the optimisation using S. cerevisiae. Were mutations induced or was the isolation procedure improved ? p.4: hairpin extension and not hairpins extension p.9: Why using capital letters for n-Dodecyl β-D-maltoside, Octyl glucoside, and 9-Amino-6-Chloro-2-Methoxyacridine ? These are simply chemicals. p.14: Please clarify "was performed using carried by" p.17: Correct spelling "Modell building

Sodium/Proton Exchanger NHE9
Corresponding author: David Drew We thank the referees for their considered evaluation. We have responded, as appropriate, to all queries below.

Winkelman et al. & Drew working in collaboration with the Beckstein and
Robinson groups have characterised horse NHE9 though a combination of methods -yeast expression, cryo-EM structure, functional assays in proteoliposomes using proton-sensitive fluorescence, biophysics of GFP-protein stability, CG-MD and homology models. The level of conclusions on ion binding sites and the putative mechanism are justified from literature and the experiments presented, and they discuss the relevance to understanding for example autism-spectrum disorders. This is fine, solid work, basically impeccable, and it shoudl interest a large readership.
Thank you! I have only few remarks: 1) the introduction. "Dysfunction of NHEs has been linked to diseases such as ....."perhaps be a bit more specific on how various diseases are either caused by NHE dysfunction, or in other cases correlated with NHE upregulation/downregulation Thank you for this suggestion. We have included more examples of NHE physiology and how they are connected to diseases.
2) the introduction: "Human disease mutations of NHE9 are linked to neurological disorders such as familial autism, ADHD and epilepsy, making NHE9 a prime drug target8-10". This remark is a bit too easy -how could a drug targeting NHE9 overcome these disorders if they are caused by NHE9 mutations and dysfunction?
We agree. This sentence is no longer included, but rather the drug targeting of NHEs is included in context of their physiological roles, which have now been explained.
3) The disordered C-terminus could perhaps be addressed a bit more except to say that it is disordered as shown for the isolated NHE1 tail (refs in the paper). Please include it in the sequence alignment (suppl. fig. 1). Could interactions occur under certain functional states that are not imposed here? It would be too much to ask for more structures that address the C-terminal interactions, but it will be highly interesting to discuss putative mechanisms of auto regulation and how they may interfere with the transport models presented here, or even disease mutations 17th Aug 2020 1st Authors' Response to Reviewers Good point. We have now included the C-terminal tail of NHE9 with the alignment in NHE1 in Figure EV1. We have clarified in the discussion that the CTD in NHE9 lacks Ca2+-CaM binding sites.
We spent about an extra year to try and obtain a cryo EM structure of NHE9 with the CTD as intact as possible and/or with known interaction partners, e.g., RACK1. Given we have no hint of any map density for the CTD, we think it is unlikely the CTD in NHE9 interacts tightly with the transporter domain in the absence of binding partners, which would disagree with the proposed auto-inhibitory mechanism in NHE1. However, since NHE9 lacks Ca2+-CaM binding sites it is possible that the CTD regulatory mechanisms have evolved differently between NHE isoforms.

Referee #2:
Proton/sodium ion exchangers (NHEs) are essential membrane transporters required to maintain the intracellular or intraorganellar pH, the sodium ion concentration and the volume. The manuscript describes the structure of a mammalian electroneutral proton/sodium ion exchanger (NHEs), namely NHE9 from horses, as determined by cryoEM. This is a major achievement. Many groups tried to achieve this goal but failed because of lack of protein production or insufficient stability of the produced protein. Key for the success of the authors was a careful comparative screening of 12 NHE9s from various vertebrates, identifying the most appropriate NHE for their studies. Apart from the structure the authors show by using mass spectrometry the importance of phosphoinositides for the dimerization of NHE9 and its activity. They use molecular dynamics simulations and related methods to investigate the mechanism. Because the methodological repertoire is very broad an appropriate evaluation of the manuscript would require experts from these various fields.
My major concerns are twofold: -The quality of the structure, as indicated by the pdb validation report is low. In particular there are many clashes of side chains, and non-hydrogen atoms outside of the density. The authors have to improve the structure or provide an explanation for its low quality.
Thank you for raising this point. NHE9 is very dynamic as can be seen by the large number of proline and glycine residues throughout the structure (Figure. EV5) and 3D variance analysis (Movie EV1). Indeed, we cannot apply C2 symmetry to improve map density, which we attribute to the mobility of the transport domains. Nevertheless, as can be seen from the Ramachandran plot, the fitting side-chains into cryo EM density and the expected RMSD deviation of NHE9 when embedded into a membrane by MD simulations, the NHE9 structure is of excellent quality for a protein determined at 3.2 to 3.5Å resolution (Table 1, Figure.  To explain, the clashes identified in the PDB validation report were predominantly located in the flexible loop regions. We have trimmed back four residues in two different loops (49-53) and (423-427); these loop regions have no bearing on the computational analysis carried out. As can be seen in the PDB validation report, the clash score decreased in NHE9* (from 48 to 6) and NHE9-CTD (from 31 to 7). The clash score is now close to average for a cryo EM structure modelled at this resolution as can be visualized in the PDB validation reports. The Ramachandran has also improved (Favored: from 84 to 94%, Allowed: from 15 to 6%) (Table 1).
-Residue D244 is considered to be essential for binding the transported sodium ion. The substantial activity of variant N243A-D244A (figure 1C) is highly surprising. This fact is not mentioned nor discussed in the text. In my opinion this observation invalidates a major part of the mechanistic discussion where D244 plays the key role for the exchange activity. I suggest to use MD simulations and binding calculations to investigate whether a sodium ion still can bind near D244A. I cannot judge the quality of the Elastic Network Modelling (ENM) and transition pathway generation.
Thank you for raising this point and, you are right, in hindsight some explanation in the text should have been included in our initial submission.
We have now made clearer in the text that the N243A-D244A mutation is part of the well-known critical ND motif, which is essential for ion-binding and transport. Our interpretation was that the NHE9 mutant was non-functional, and yet we did not explain why this ND mutant did not completely abolish the pH induced response by Na+ addition as expected. Nevertheless, since Na+/H+ antiporters have a welldocumented ion-binding site (as can also be seen here by bioinformatic analysis and by structural comparison to crystal structures, especially Tl+-bound PaNhaP), the ionbindings site location and the role of the strictly conserved ion-binding aspartate for Na+ coordination were not in doubt i.e., as such, the fact that we could still see a measurable response had no bearing on the final mechanism, see figure below.

Figure showing the MD simulations of Na+ binding to NHE9 (left and center panel) and the corresponding location of the Tl+ bound site in the bacterial homologue PaNhaP (right panel)
We should point out that in MD simulations when D244 was protonated we saw 0% Na + binding (Appendix Table 2). We have made this control MD simulations clearer to the reader by including a new figure Appendix Figure S5; also pasted below. Either empty liposomes or a known dead-mutant, should have been appropriate controls in a proteoliposome transport assays. However, since our initial submission we have concluded that these "negative" controls inaccurately assessed the background signal in the transport assay. This became apparent to us when we could still measure the same Na + -induced response for a completely unrelated protein, the rat fructose transporter GLUT5. For sake of clarity, we now only show the response in the assay to rat GLUT5, rather than either empty liposomes or the NapA dead mutant ( Figure 1C). We further have included a Na + -dependent titration of rat GLUT5 and N243A-D244A mutant, to show that the signal-to-noise is yet high enough to measure NHE9 kinetics; calculated from 3 independent protein reconstitutions ( Figure  1D); pasted below. Purified rat GLUT5, like NHE9, has poor stability in detergent as compared to bacterial NapA. Our current theory is that during detergent removal and reconstitution into liposomes a fraction of the mammalian proteins aggregate, which effects the integrity of some ATP-containing liposomes. We think that under a pH gradient these liposomes burst upon NaCl addition due to osmotic pressure, which causes a proton leak and an increase in the background signal compared to empty liposomes or the dead NapA mutant. Though not ideal, we have spent considerable effort on optimizing the NHE9 proteoliposome assay, and it is unlikely we can resolve this issue in a short time frame. To put this work into broader context, however, NHEs lack working protocols using purified components and, in our hands, the functional assay development has been of equal effort as the cryo EM work. Notably. in this case, NHE9 activities are challenging to record in vivo due to their endosomal location.
Minor concerns: p.3 and methods: Please explain the optimisation using S. cerevisiae. Were mutations induced or was the isolation procedure improved ?
Yes, we made a small C-terminal truncation based on a region of predicted disorder as described in the beginning of the methods section.
Thank you, these have now been updated. p.14: Please clarify "was performed using carried by" Thank you, we have now fixed this typo.
p.17: Correct spelling "Modell building Thank you, we have now fixed this typo.

7th Sep 2020 1st Revision -Editorial Decision
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The authors have addressed my previous criticism satisfactorily: the quality of the structure has been improved considerably. A satisfactory explanation for the "activity" of the double mutant has been provided, new experiments have been performed demonstrating that the observed "activity" of the double mutant was an experimental artifact. The minor corrections have been done.

9th Sep 2020 2nd Authors' Response to Reviewers
The authors performed the requested editorial changes.

10th Sep 2020 2nd Revision -Editorial Decision
Dear Dr Drew, Thank you for submitting the revised version of your manuscript. I have now evaluated your amended manuscript and concluded that the remaining minor concerns have been sufficiently addressed.
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