Author response:
The following is the authors’ response to the original reviews.
eLife assessment
The study elucidates a detailed molecular mechanism of the initial stages of transport in a medically relevant GABA neurotransmitter transporter GAT1 and thus generates useful new insights for this protein family. In particular, it presents convincing evidence for the presence of a "staging binding site" that locally concentrates Na+ ions to increase transport activity, whilst solid evidence for how Na+ binding affects the larger scale dynamics.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
The manuscript authored by Stockner and colleagues delves into the molecular simulations of Na+ binding pathway and the ionic interactions at the two known sodium binding sites site 1 and site 2. They further identify a patch of two acidic residues in TM6 that seemingly populate the Na+ ions prior to entry into the vestibule. These results highlight the importance of studying the ion-entry pathways through computational approaches and the authors also validate some of their findings through experimental work. They observe that sodium site 1 binding is stabilized by the presence of the substrate in the S1 site and this is particularly vital as the GABA carboxylate is involved in coordinating the Na+ ion unlike other monoamine transporters and binding of sodium to the Na2 site stabilizes the conformation of the GAT1 by reducing flexibility among the helical bundles involved in alternating access.
Strengths:
The study displays results that are generally consistent with available information from experiments on SLC6 transporters particularly GAT1 and puts forth the importance of this added patch of residues in the extracellular vestibule that could be of importance to the ion permeation in SLC6 transporters. This is a nicely performed study and could be improved if the authors could comment on and fix the following queries.
We thank our reviewer for the overall positive evaluation.
Weaknesses:
(1) How conserved are the residue pair of D281-E283 in other SLC6 transporters. The authors commented on the presence of these residues in SERT but it would be nice to know how widespread these residues are in other SLC6 transporters like NET, GlyT, and DAT.
We have created a sequence alignment of the entire human SLC6 family (Supplementary Figure 1) and found that E283 is polar or charged in all SLC6 transporters. D281 shows a higher level of conservation across the family compared to E283. D281 is negatively charged in approximately 50% of the SLC6 family members, an aspartate in all GABA transporters and a glutamate in all monoamine transporters.
(2) Further, one would like to see the effect of individual mutations D281A and E283A on transport, surface expression, and EC50 of Na+ to gauge the effect on transport.
We have carried out experiments to investigate the effects of the individual mutations. The results revealed intermediate effects between WT and the double mutant (D281A-E283A) and showed that the effects mostly align with the degree of conservation, as a neutralisation of D281 by alanine has a stronger effect than the E283A mutant. Both single mutants had minimal effects on the sodium dependence of uptake, D281A had a stronger effect on expression, Km and Vmax as compared to E283. Only D281A reduced surface expression, while E283A expresses to a similar level as wild type GAT1.
(3) A clear figure of the S1 site where Na+ tends to stay prior to Na1 site interactions needs to be provided with a clear figure. Further, it is not entirely clear how access to S1 is altered if the transporter is in an outwardoccluded conformation if F294 is blocking solvent access. Please comment.
We have modified the structural images in Figure 1, 5, 6 and 7 to improve their comprehensibility. We have also added a comment on the role of F294 as part of the outer hydrophobic gate to the discussion. In short, F294 does not occlude the passage to the S1 as long as GAT1 is outward open, and we find that GAT1 is outward open in all sodium binding simulations.
(4) The p-value of the EC50 differences between GAT1WT and GAT1double mutant need to be mentioned. The difference in sodium dependence EC50 seems less than twofold, and it would be useful to mention how critical the role of the recruitment site is. Since the transport is not affected the site could play a transient role in attracting ions.
We have added p-values or standard deviation to our data.
(5) It would be very nice to know how K+ ions are attracted by this recruitment site. This could further act as a control simulation to test the preference for Na+ ions among SLC6 members.
We think that attraction of potassium to the recruitment site is not of relevance, as the residues are at the extracellular side and exposed to bulk, where the concentration of sodium is high (typically 130-150 mM), while the concentration of potassium is very small (3-5 mM). Exploring sodium binding by simulations for all SLC6 members could be interesting, but clearly outside the scope of this manuscript.
(6) Some of the important figures are not very clear. For instance, there should be a zoomed-in view of the recruitment site. The current one in Fig. 1b and 1c could be made clearer. Similarly as mentioned earlier the Na residence at the S1 site away from the Na1 and Na2 sites needs to be shown with greater clarity by putting side chain information in Fig. 6d.
We have modified the structural images in Figure 1, 5, 6 and 7 to improve their comprehensibility.
(7) The structural features that comprise the two principal components PC1 and PC2 should be described in greater detail.
We have modified Figure 6 and added images that show the motions along PC1 and PC2. In addition, these are now better explained in the text.
Reviewer #2 (Public Review):
Summary:
Starting from an AlphaFold2 model of the outward-facing conformation of the GAT1 transporter, the authors primarily use state-of-the-art MD simulations to dissect the role of the two Na+ ions that are known to be cotransported with the substrate, GABA (and a co-transported Cl- ion). The simulations indicated that Na+ binding to OF GAT depends on the electrostatic environment. The authors identify an extracellular recruiting site including residues D281 and E283 which they hypothesized to increase transport by locally increasing the available Na+ concentration and thus increasing binding of Na+ to the canonical binding sites NA1 and NA2. The charge-neutralizing double mutant D281A-E283A showed decreased binding in simulations. The authors performed GABA uptake experiments and whole-cell patch clamp experiments that taken together validated the hypothesis that the Na+ staging site is important for transport due to its role in pulling in Na+.
Detailed analysis of the MD simulations indicated that Na+ binding to NA2 has multiple structural effects: The binding site becomes more compact (reminiscent of induced fit binding) and there is some evidence that it stabilizes the outward-facing conformation.
Binding to NA1 appears to require the presence of the substrate, GABA, whose carboxylate moiety participates in Na+ binding; thus the simulations predict cooperativity between binding of GABA and Na+ binding to NA1.
Strengths:
- MD simulations were used to propose a hypothesis (the existence of the staging Na+ site) and then tested with a mutant in simulations AND in experiments. This is an excellent use of simulations in combination with experiments.
- A large number of repeat MD simulations are generally able to provide a consistent picture of Na+ binding. Simulations are performed according to current best practices and different analyses illuminate the details of the molecular process from different angles.
- The role of GABA in cooperatively stabilizing Na+ binding to the NA1 site looks convincing and intriguing.
We thank the review for the very supportive assessment.
Weaknesses:
- Assessing the effects of Na+ binding on the large-scale motions of the transporter is more speculative because the PCA does not clearly cover all of the conformational space and the use of an AlphaFold2 model may have introduced structural inconsistencies. For example, it is not clear if movements of the inner gate are due to an AF2 model that's not well packed or really a feature of the open outward conformation.
The long range effect of sodium binding to GAT1 and destabilisation of the inner gate has, based on our data, a causal effect. PCA separates conformational motions into degrees of freedom and sorts them according to the largest motions. Motions of TM5a were among the 2 largest motions, which suggests that these are relevant motions. To directly quantify their behaviour, we measured informative distances at the inner gate of GAT1, as shown in Figure 6i,j,k and separated data according to the presence of sodium in NA2.
For the following reasons we exclude that the results are a consequence of structural inconsistencies introduced by AlphaFold2 and therefore not reflecting functionally relevant effects:
(1) If depending on the model instead of sodium binding, the effects should not be correlated with the presence of sodium in the NA2 binding site.
(2) We carried out new simulations starting from the occluded GAT1 structure (Figure 6j,k). The data shows that in the occluded state the distance across the inner vestibule and the length of TM5a differ, consistent with our interpretation of the data. As sodium binding fixes GAT1 outwardfacing, as it also occurs in other SLC6 family members (Szöllősi and Stockner, 2022), the distances of the outward-open GAT1 are at the short extreme of the scale, distances of the inward-open state of the cryo-EM structure(s) are at the other extreme, while the occluded conformation of GAT1 shows intermediate values.
(3) We have observed the same property in SERT, for which we used experimental structures as starting structure (Gradisch et al., 2024), suggesting that this could be a generally mechanism.
(4) All available structures from the entire SLC6 family are consistent with structural effects of TM5a in response to bundle domain motions and therefore to binding of sodium to NA2 as it stabilized the outward-open state as well as transition to the inward facing conformation.
- Quantitative analyses are difficult with the existing data; for example, the tICA "free energy" landscape is probably not converged because unbinding events haven't been observed.
Simulations can always be too short and therefore not fully describe the complete underlying conformational ensemble. We added a statement in the discussion indicating this shortcoming. With respect to the tICA analysis in our manuscript, the tICA approach does, by design, not need long simulations that capture the full binding and unbinding in multiple instances to construct a correct free energy landscape. Instead, the tICA method builds on Markov chain dependencies and relies only on the convergence of transitions between hundreds of conformational microstates and the fluxes between them. The free energy profile derived for the S1, including NA1, TMP and NA2 and up to the salt bridge of the outer gate is well converged and we observed many transitions. In contrast, the entry from the recruitment side to the S1 has most likely a too low density of microstate and a too small number of transition to be considered converged with respect to quantifying the free energy of binding from bulk. We now explain this shortcoming.
Recommendations for the authors:
Reviewer #1 (Recommendations for The Authors):
Authors should furnish p-values in the figure legends for experimental results.
We have added the p-values to text and figure legends.
Reviewer #2 (Recommendations For The Authors):
- Deposit simulation data in a public repository (input files, trajectories (possibly subsampled)).
We deposited the data to Zenodo and provided the DOI: 10.5281/zenodo.10686813 to the data. As we were unable to upload the trajectories to zenodo, we deposited the starting and the end structures of the simulations.
- Please include a short discussion of the reliability of using an AF2 model instead of experimental structures. What is expected to be correct/which parts of the structure are potentially incorrect? What makes you think that the AF2 model is a good model of the OF conformation of GAT1?
Unfortunately, an outward-facing structure of GAT1 is not available. We have initially worked with an outward-open homology model of GAT1 based on SERT (build with MODELLER), but the structural differences between SERT and GAT1 are sufficiently large that these models did not behave well in simulations and too frequently could not maintain a sealed inner gate, also forming a channel. In contrast to the SERT-based GAT1 model, the AlphaFold2 model of GAT1 behaved as expected and consistent with the behaviour of SERT in simulations and with general knowledge of protein dynamics from literature. Based on structural analysis of our simulations and on the comparison to SERT we could not identify a region of GAT1 which would be potentially behave incorrect or unexpectedly. We added a statement to the discussion on this potential limitation of the use of homology models.
- Fig 1a: Na+ densities are not very clear (both due to small size and the transparency). I have a hard time seeing where bulk, 2*bulk regions are --- are you showing "onion shells" of density? Perhaps investigate presenting as cuts through the full density?
I like the labelling in terms of absolute density and multiples of bulk.
We have created new images to improve the visualisation of data. The data are shown as onion shells (isosurface), with the shells at the indicated densities. This is now clearly stated. Transparency is needed, otherwise e.g. the inner onion shells would not be visible. The cut-through is intuitive, but we could not find a useful plain, as the densities are too extensively distributed in 3D and not on a single plain.
- Fig 1h-k: would be clearer if "recruitment site" (TMP?) was indicated in the figure.
We have created a new image for the recruiting site (Figure 1b,c) and temporary site (Figure 1g) and indicated these two sites as appropriate.
- Show time series of Na+ binding with a suitable order parameter (z or distances to NA1 and NA2?) to show how ions bind spontaneously. Mark the different sites. Mark pre- and post-binding parts of trajectories.
We have added time series for every simulation that shows sodium binding to the NA1 or NA2 to the supplementary information Figure 2a,b,c. These quantify the distances to the recruiting site, the temporary site and the respective sodium binding site.
- PCA - how much of the total variance was captured by PC1 and PC2?
The variance captured by the PCs are shown as eigenvalues in supplementary information Figure 4. PC1 captures about 19% of the variance, PC2 8%.
- "We found that the inner hydrophobic gate is dynamic in the absence of Na2" -- is this instability due to the AF2 model or likely realistic? E.g. was similar behaviour ever observed in simulations of the occluded state?
In simulations of the occluded state we do not see such instabilities as observed in the outward-open state in the absence of sodium (Figure 6). As these larger scale fluctuations are not randomly distributed across all simulations starting from the AlphaFold2 models, but confined to the systems without sodium, it is unlikely an effect of the AlphaFold2 model.
Please note, we have seen comparable behaviour in simulations of SERT starting from experimental structures (Gradisch et al., 2024), therefore suggesting a more general mechanism.
- Cooperativity between GABA-binding and Na+ binding to NA1: How would this lead to an experimentally measurable signature, i.e., which experiments could validate this interesting prediction?
Direct detection of cooperativity is difficult to separate from other effects in experiments, as sodium binding and transport involves NA1 and NA2, NA2 has a higher affinity according to our data, while mutations will not only affect cooperativity, but will also have other effects.
Conformational changes can also complicate experimental detection, as NA2 stabilises the outward-open conformation, while NA1+GABA binding triggers the transition to the inward-open state. To quantify cooperativity, it would be important to isolate the cooperative from all other effects, which is a challenge. Support for cooperativity has been found by (Zhou, Zomot and Kanner, 2006; Meinild and Forster, 2012) using this route. In the first paper the authors make use of lithium that only binds to the NA2, even though lithium is not only a mere NA2 selective ligand and otherwise identical to sodium. By comparing two GABA concentrates the authors showed that the sodium dependence of GABA transport is left shifted at higher GABA concentrations, which is not the case in the absence of lithium. This data is indirect, but consistent with cooperativity between GABA and NA1-bound sodium, as GABA transport mainly reflects binding of sodium to NA1. Similar approaches could be further explored, for example by varying the GABA concentration instead of sodium. Other options could be to create an outward-facing and conformationally locked GAT1 and to measure the cooperativity of sodium and GABA binding using for example the scintillation proximity assay. Most likely the assay would also need a way to be NA2 binding independent. We are not aware of such a GABA transporter system.
- There are some instances of [SI Figure] or [citation needed] that should be cleaned up.
We have corrected these instances.
References
Gradisch, R. et al. (2024) ‘Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors’, Nature Communications, 15(1), p. 417. Available at: https://doi.org/10.1038/s41467-023-44637-6.
Meinild, A.-K. and Forster, I.C. (2012) ‘Using lithium to probe sequential cation interactions with GAT1’, American Journal of Physiology. Cell Physiology, 302(11), pp. C1661-1675. Available at: https://doi.org/10.1152/ajpcell.00446.2011.
Szöllősi, D. and Stockner, T. (2022) ‘Sodium Binding Stabilizes the Outward-Open State of SERT by Limiting Bundle Domain Motions’, Cells, 11(2), p. 255. Available at: https://doi.org/10.3390/cells11020255.
Zhou, Y., Zomot, E. and Kanner, B.I. (2006) ‘Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1’, The Journal of Biological Chemistry, 281(31), pp. 22092–22099. Available at: https://doi.org/10.1074/jbc.M602319200.
