SI Appendix

Authors' response (23 March 2023)
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GENERAL ASSESSMENT
Yang et al. present valuable insight about ligand interaction with SERT. It is notable for the use of endogenously expressed SERT from pig brain, rather than from a heterologous expression system, and the value of using Fab fragments that as a tool for detecting and purifying SERT. The use of natively expressed SERT allows insight into binding sites for endogenous membrane components, including lipids, that copurify with the transporter. Data on binding of the psychostimulants methamphetamine and cocaine to the purified protein also adds to our knowledge of substrate and inhibitor interactions with SERT.
In general, we liked the manuscript but have some suggestions for improvement and clarification. There was unanimous agreement that the conclusions regarding SERT oligomerization were stated in a way that many readers would mis-interpret. Although the manuscript states that the result of the study do not exclude SERT oligomerization in its native environment, that statement is not reflected in the abstract and is downplayed in the discussion. The discussion clearly points out why some previous proposals for SERT oligomerization may conflict with our knowledge of SERT structure and function. However, it is also clear that the extraction of SERT using DDM, although it's influence on SERT structure may be mild, can remove other membrane components that are required for oligomer stability.
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We have qualified our statements related to SERT oligomerization to make it clear that we are studying SERT extracted using a mild non ionic detergent and, in the membrane and in the presence of specific lipids, the oligomerization state of SERT might be different.
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A second issue raised by the reviewers concerns the poses of bound ligands and their effect on conformation. There is an expectation that substrate binding converts NSS transporters like SERT to a more inward-facing conformational ensemble while some inhibitors (particularly cocaine in the case of SERT) have the opposite effect. Statements in the manuscript that METH stabilizes an outward-open conformation while cocaine stabilizes an occluded state seem to contradict this expectation. A close reading of the results, and examination of the structures, indicates that the backbone of SERT is outward-open in both cases and that the orientation of Phe372 occludes cocaine but not METH. Thick and thin gates have already been defined for LeuT so the implication that orientation of Phe372 represents an additional gating process may confuse readers.
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We have rephrased our description of the conformations of the SERT-ligand complexes such that they are in better alignment with previous descriptions of transporter conformations and the nature of the gates.
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The observation that METH does not stabilize a less-outward-open conformation may result from its lower affinity for SERT and one wonders why a more SERT-selective amphetamine derivative such as MDMA or p-chloroamphetamine was not used. Was the objective to compare the binding pose and conformational response of SERT to that of dDAT with the same ligands?
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Yes, we carried out studies on pSERT to allow the most 'parallel' comparisons with the previously studied dDAT complexes.
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A third issue is the designation of SERT purified from pig brain as nSERT rather than "native porcine SERT" (pSERT or ssSERT for Sus scrofa). One wouldn't use nSERT to define SERT extracted from Drosophila or C. Elegans by the same technique.
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We have defined the transporter as pSERT in the revised version of the manuscript.
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RECOMMENDATIONS
Revisions essential for endorsement by Biophysics Colab:
<ol>
<li>Clearly indicate that the lack of oligomer detection in detergent-solubilized SERT is not evidence against SERT oligomerization in situ, and that detergent can disrupt interactions required for oligomerization, thereby biasing the oligomeric status of SERT. Also, use an alternative for DDM-solubilized SERT other than "native" or "nSERT"</li>
</ol>
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See above
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<ol>
<li>Distinguish between the local occlusion of cocaine by reorientation of the Phe-372 side chain and the occluded conformational state of SERT that involves movement of TMs 1 and 6 towards TMs 8 and 10. Also explain the choice of METH rather than an amphetamine derivative more selective for SERT. Do the structures explain the difference in METH affinity between SERT and dDAT?</li>
</ol>
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Also see above
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<ol>
<li>Because neither the DHA density nor the MD simulations provide an unambiguous identification of DHA, designation of this density as DHA should be clearly stated as provisional.</li>
</ol>
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We have repeated the MD simulations, also exploring occupancy of the allosteric site by DDM, and find that DDM is preferred over DHA. We have 'toned down' our suggestions that DHA might occupy the site accordingly.
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Additional suggestions for the authors to consider:
<ol>
<li>Does DHA fulfill a functional role? The carboxyl group is close to Arg141, which can form an ion pair with Glu531 to close the extracellular pathway. Is there any indication that DHA could interfere with this process, and alter SERT activity?</li>
<li>Is there sufficient purified material to perform a lipidomic analysis and to confirm the identity of DHA at the allosteric site?</li>
<li>Would incubation of the membranes with METH or cocaine prior to detergent extraction affect the composition of associated lipids?</li>
<li>PIP2 has been proposed to promote SERT association in vivo. Is it possible that PIP2 addition would change the oligomeric nature of DDM-extracted SERT?</li>
<li>We suggest trying to polish the final particle stack of both data sets further. Have the authors tried to separately refine the 3D classes from Relion, and not to combine them? Alternatively, one could perform an additional round(s) of heterogenous refinement on the final particle stack, and a final nonuniform refinement. There seems to be an opportunity to improve the quality of maps by further polishing the particles.</li>
</ol>
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(This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

Evaluation statement (24 May 2023)
Yang et al. present valuable information about ligand interactions with the serotonin transporter SERT, innovatively purified from pig brain using Fab fragments. The approach of using natively expressed SERT is notable for its potential insight into binding of endogenous membrane components such as lipids. Data distinguishing binding of the psychostimulants methamphetamine and cocaine add to our knowledge of substrate and inhibitor interactions with SERT and allow direct comparison with the closely related dopamine transporter DAT. The authors carefully state the limitations of their findings, including the possibility that the monomeric transporter stable in detergent micelles might exist in a multimeric state in native membranes.
Biophysics Colab considers this to be a convincing study and recommends it to scientists interested in the structure, mechanism and ligand interactions of neurotransmitter transporters.
(This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants

Figures and data

Purification and biochemical analysis of pSERT from native membranes.

The cryo-EM structure of pSERT in complex with (+)-methamphetamine or cocaine, respectively.

Ligands occupy the central site.

Cholesteryl hemisuccinate (CHS) and DDM binding in SERT.