Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation

Reviewer #1 (Public review):

The authors have tried to identify the plausible Na+ entry pathway in an important SLC6 member GAT1, using computational approaches to assess residence times of the ions as they enter the vestibule of GAT1. The authors identify a patch of negative residues in TM6a and implicate them for being important to attract the Na+ ions during their movement towards the binding sites Na1 and Na2. Besides this they also suggest that sodium binding at site 1 is flexible and at times can occupy the primary binding site when the substrate is not available. Na2 site as other literature also suggests is demonstrated to be vital for the stability of the outward-open state.

Studies of ion permeation are challenging given that the states are challenging to trap through structural studies and computational methods are vital for understanding these steps. The authors suggest that two negatively charged residues are vital to attract Na+ ions to the vestibule. Using a combination of simulations and PCA analysis the authors identify the importance of Na+ binding at site 2 that stabilises the outward-open state and the flexibility observed in Na1 site for ion binding which happens alongside substrate in the GABA bound state. The study reconfirms earlier observations in the SLC6 family that Na2 site is critical for conformational transitions and Na1 site is substrate dependent in amino acid transporters.
One of the challenges in such studies is to conclusively establish the presence of additional Na+ sites or regions of ion-binding with experimental structures as they are nearly impossible to trap. Such studies using simulations therefore become the only resort to understand such phenomena.

The work is likely to further provide insights into the transport mechanism of GAT1 and lends credence to some structural studies where the sodium at site1 is displaced but the ion remains proximal to the bound substrate.

Author response:

The following is the authors’ response to the previous reviews.

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 the reviewer for the overall positive assessment of our work.

Comments on revised version:

The authors have satisfactorily addressed my comments and this has significantly improved the clarity of the manuscript.

The only point that I would like to inquire about is the role of EL4 in modulating Na+ entry.

In the simulations do the authors see no role of EL4 in controlling Na+ entry. It is particularly intriguing as some studies in the recent past displayed charged mutations in EL4 of dDAT, SERT and GAT1 as being detrimental for substrate entry/uptake. It would therefore be nice to add a small discussion if there is any role for EL4 in Na+ entry.

In this study we focused on sodium binding to the sodium binding site NA1 and NA2 and discovered the role of negatively charged residues at the beginning of TM6 contribution to sodium binding. Our data shows less than average interactions of sodium ions with EL4. In particular, we do also not observe any prominent role for D355, which is the only negatively charged residues in EL4a. We associate this effect to the presence of four positively charged residues (R69,Y76, K350, R351) surrounded D355 and an electrostatic repulsion by a local positive field, which is also visible in Figure 1k. Following the suggestion of the reviewer, we added a short statement to the last paragraph of the discussion.

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 co-transported with the substrate, GABA (and a cotransported 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 D281AE283A 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 reviewer for the overall positive assessment of our work.

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 a AF2 model that's not well packed or really a feature of the open outward conformation.

We do not think that the results of the manuscript and in particular the large scale motions are speculative or dependent too much on the limitations of PCA. We only use PCA for Figure 6a-d,6g,h. Motions of SLC6 transporters (and of any other transporter) are much more complex than a single 2D PCA plot could every capture. We therefore used PCA here only to identify the two motions with the largest amplitude, show in Figure 6a-d, 6g,h.

Given that all the ~13000 degrees of freedom of GAT1 contribute to conformational differences, a dimensionally reduction method like PCA can be very helpful for extracting dominant motions. Structure comparison showed that motions observed in PC1 captured a large portion of the motions of occlusion (Figure 6c,d) when compared to the full transition observed in the unfiltered trajectories (See Figure 6e,f). PCA therefore helps to extract this main motions.

For completeness, we show a series of structures from the unfiltered trajectories in figure 6e,f. In the overlay, the motion of occlusion is more difficult to observe, because convoluted with all other degrees of freedom. In figure 6e,f, the structures are aligned with the maximum likelihood method theseus, while the coloring is based on the amplitudes measured by PCA to visualize the regions moving relative to each other with largest amplitude. All other structural measures, including the opening of the inner gate (Figure 6i-k), are direct measures of the raw trajectories.

With respect to the question of the instability of the inner gate, we made similar observations for hSERT (please see DOI: 10.1038/s41467-023-44637-6) using the experimentally determined structure as starting point. We find a weakening of the inner gate for sodium free SERT and at intermediate or full occlusion of sodium- and serotonin-bound SERT. These previous data on SERT corroborate our finding and indicates that the effect could be a general feature of the SLC6 transporter family.

Unfortunately no outward-open structure of GAT1 was available for this study. AlphaFold2 models have limitations and we are well aware of these limitations, but AlphaFold2 can also make high quality models including small adjustment of backbone positions, if the sequence identity is high, as in the current project (43% sequence identity for the transmembrane region). For GAT1 (as described in the manuscript) we initially tested hSERT based model created with MODELLER. MODELLER uses as premises the assumption that the protein backbone does not change or only very little between the template protein and the target protein. These MODELLER created models did not perform well, because of a slight shift in the position of the backbone, which is a consequence of consistently smaller side chains in the bundle domain-scaffold domain interface of GAT1 as compared to SERT.

In the simulations described in the manuscript (using the AlphaFold created model) we observed that the overall structural and dynamic parameters and in particular also observation at the inner gate are very similar to the results described in our papers on sodium binding to SERT using experimental SERT structures. The differences of Na1 binding are explained in the manuscript and are contingent to the residue difference of D98 in SERT and the corresponding residue G65 in GAT1. This makes us confident about the quality of the obtained data. Please see DOI: 10.3390/cells11020255; DOI: 10.3389/fncel.2021.673782.

- 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.

The tICA analysis is a Marco State Model approach, which relies on the convergence of transitions between a large number of microstates. A limited number of trajectories showing full sodium unbinding are not obligatory for converged dataset, but the transitions between the microstates must to be converged. For the transitions within the S1 we have many transitions and very good convergence for transition probabilities within the S1. We limit interpretation of free energy data and discussion on this part of the free energy surface. The supporting information (Figure S5) reports on the quality of the tICA analysis. Flat lines with a time lag larger than 40 ns is consistent with a converged model based on the data of the trajectories used for the analysis, and consistently, also the Chapman-Kolmogorov tests show minimal difference between estimates and predictions.

We see about 40 binding event from the extracellular side to the S1, which seems insufficient for a converged quantification for sodium transiting from the extracellular side to the S1. We state this limitation of the dataset in the results section of the manuscript.