Structural basis for recognition of diverse antidepressants by the human serotonin transporter

Coleman, Jonathan A.; Gouaux, Eric

doi:10.1038/s41594-018-0026-8

Cited by 133 publications

(150 citation statements)

References 50 publications

(57 reference statements)

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“…To facilitate 5HT reuptake, the transporter needs to undergo distinct conformational changes. In principle, these changes expose the substrate binding site(s) to one side of the membrane at a time, according to the so-called alternatingaccess mechanism [9], support for which has been provided by structural modeling and biochemical experiments [10][11][12], and recently also by X-ray and cryo-EM crystallography [13][14][15]. Taken together, the hSERT transport cycle can be represented roughly as shown in Fig 1, albeit with the caveat that several important details remain elusive.…”

Section: Introductionmentioning

confidence: 90%

A structural model of the human serotonin transporter in an outward-occluded state

Hellsberg

Ecker

Stary-Weinzinger

et al. 2019

Preprint

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The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin.Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

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Section: Introductionmentioning

confidence: 90%

A structural model of the human serotonin transporter in an outward-occluded state

Hellsberg

Ecker

Stary-Weinzinger

et al. 2019

Preprint

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“…Selective serotonin reuptake inhibitors (SSRIs) are a class of drugs which inhibit SERT and are used to treat major depression and anxiety disorders 7 . Using x-ray crystallography and cryo-EM, we have determined structures of thermostabilized variants of human SERT complexed with SSRIs, which together explain many of the common features and differences associated with SERT-SSRI interactions 8,9 . SSRIs are competitive inhibitors that bind with high-affinity and specificity to a central substrate-binding site in SERT, preventing 5-HT binding and arresting SERT in an outward-open conformation 2,3,9 . The central site in NSSs is composed of three subsites: A, B, and C 10 (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…The heterocyclic electronegative group of the ligand is positioned in subsite B 5 . For example, the alkoxyphenoxy groups of reboxetine and nisoxetine 11 in Drosophila DAT (dDAT) structures, the halophenyl groups of cocaine analogs in dDAT and Scitalopram in SERT, and the catechol derivatives in DCP-dDAT and sertraline-SERT all occupy subsite B 8,9,12 . In addition to the central binding site, the activity of SERT and NSSs can also be modulated by small-molecules which bind to an allosteric site located in an extracellular vestibule, typically resulting in non-competitive inhibition of transport 9,[13][14][15] .…”

Section: Introductionmentioning

confidence: 99%

“…Despite these efforts, however, the binding pose of paroxetine remains a subject of debate 8,9,[18][19][20] .…”

Section: Introductionmentioning

confidence: 99%

“…1b). X-ray structures of the SERT-paroxetine complex revealed that the piperidine ring binds to subsite A while the benzodioxol and fluorophenyl groups occupy subsite B and C in the central site, respectively 8,9 (ABC pose). However, recent mutagenesis, molecular dynamics, and binding studies with paroxetine analogues suggest that paroxetine may occupy two distinct poses in which the benzodioxol and fluorophenyl groups reside in subsite B or C, depending on the rotameric position of Phe341 and the presence of the thermostabilizing mutation Thr439Ser 18,20 (ACB pose, Fig.…”

Section: Introductionmentioning

confidence: 99%

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Chemical and structural investigation of the paroxetine-human serotonin transporter complex

Coleman

Navratna

Antermite

et al. 2020

Preprint

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Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake.Structural and biochemical studies aiming to understand the binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation. Such modifications to SERT have led to questions about the relationships of these structures to the bona fide conformation and inhibitor binding poses of the wild-type transporter. To address these concerns, we characterized wildtype SERT with truncated N-and C-termini and thermostabilized variants of SERT bound with paroxetine using x-ray crystallography, single particle cryo-EM and biochemical techniques.Moreover, using a C-H functionalization approach to synthesize enantiopure analogues, we replaced the halide of the fluorophenyl group in paroxetine with either bromine or iodine. We then exploited the anomalous scattering of Br and I to define the pose of the respective paroxetine analog. These structures provide mutually consistent insights into how paroxetine and its analogs bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

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Transfer of stabilising mutations between different secondary active transporter families

et al. 2021

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Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid‐xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single‐point mutation, G411V, trapping the protein in the inward‐facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.

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Structural basis for recognition of diverse antidepressants by the human serotonin transporter

Cited by 133 publications

References 50 publications

A structural model of the human serotonin transporter in an outward-occluded state

A structural model of the human serotonin transporter in an outward-occluded state

Chemical and structural investigation of the paroxetine-human serotonin transporter complex

Transfer of stabilising mutations between different secondary active transporter families

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