Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport
This study investigated serotonin transporter (SERT) complexes with ibogaine to illustrate structure-based mechanisms for transport in serotonin transporter (SERT). The investigation reported that cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, closed and inward-open conformations with ibogaine binding to the central binding site, and the closing extracellular gate with movements of TMs 1b and 6a. The intracellular gate opening had a hinge-like movement of TM1a and the partial unwinding of TM5 that together built a permeation pathway enabling substrate and ion diffusion to the cytoplasm. These structures show the structural rearrangements which occur from the outward-open to inward-open conformations, and give an important insight into the working mechanism of neurotransmitter transport and ibogaine inhibition.
Authors
- Coleman, J. A.
- Gouaux, E.
- Tajkhorshid, E.
Published
Abstract
The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.
Research Summary of 'Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport'
Introduction
The serotonin transporter (SERT) is a sodium- and chloride-dependent membrane protein that terminates serotonergic signalling by recapturing serotonin into presynaptic neurons. SERT belongs to the neurotransmitter sodium symporter (NSS) family alongside the dopamine and noradrenaline transporters; these transporters are targets for antidepressant drugs (selective serotonin reuptake inhibitors) and are modulated by psychostimulants such as cocaine and amphetamines. Although structural studies of bacterial NSS homologues (for example LeuT) have outlined large-scale transmembrane helix movements during the transport cycle, the conformational changes that underlie transport and inhibition in eukaryotic SERT remained incompletely characterised. Coleman and colleagues set out to define structure-based mechanisms of transport and inhibition in human SERT by determining cryo-electron microscopy (cryo-EM) structures of SERT bound to ibogaine, a psychoactive alkaloid reported to be a non-competitive inhibitor of SERT. The study aimed to capture SERT in multiple transport-related conformations, locate the ibogaine-binding site, characterise ligand interactions, and combine structural data with biochemical, mutational and computational approaches (docking and molecular dynamics) to explain how ibogaine inhibits transport and how SERT transitions between outward-open, occluded and inward-open states.
Methods
The investigators used a combination of biochemical characterisation, radioligand binding and uptake assays, single-particle cryo-EM, mutagenesis, labelling in nanodiscs, computational docking and molecular dynamics (MD) simulations. Multiple SERT constructs were employed: an N- and C-terminally truncated wild-type (ΔN72/ΔC13), a thermostable ts2-active construct (retaining transport activity), a ts2-inactive variant locked in an outward-open conformation, and a C7x background with fewer reactive cysteines. Antibody fragments (15B8 Fab and an 8B6 scFv) were used to increase particle mass and aid cryo-EM reconstruction; some Fab combinations preserved transport activity while others rendered the complex transport-inactive. Functional characterisation included 5-HT uptake assays in HEK-293S GnTI- cells (Michaelis–Menten analysis) and radioligand binding by scintillation proximity assays (SPA) using [3H]paroxetine or [3H]ibogaine. A cysteine-reactivity labelling assay (S277C mutant) in nanodiscs probed accessibility of the cytoplasmic permeation pathway. Binding assays were performed in buffers containing NaCl, KCl or NMDG-Cl to test ion-dependence. For structural work, SERT–antibody complexes were purified in the presence of paroxetine, ibogaine or noribogaine and vitrified for cryo-EM. Data were collected on 200–300 kV microscopes with direct electron detectors and processed using MotionCor2, Gctf, DoG-Picker, cryoSPARC, RELION, cisTEM and PHENIX. Reconstructions were obtained for outward-open, occluded and inward-open conformations at nominal resolutions ranging from ~3.6 Å to ~4.2 Å (noribogaine reconstruction at ~6.3 Å). Models were built by rigid-body fitting of available X-ray structures followed by iterative rebuilding in Rosetta, Coot and PHENIX, with cross-validation against half maps. Computational approaches comprised an extensive docking workflow that exhaustively sampled ibogaine poses in outward-open, occluded and inward-open SERT conformations (millions of initial placements, clustering by r.m.s.d., energy filtering and selection by pair interaction energies and cross-correlation with cryo-EM density). Selected poses were refined by MD simulations in explicit POPC bilayers with NaCl or KCl (~100 mM), using CHARMM force fields and force-field parameters for protonated ibogaine derived and optimised from CGenFF and quantum calculations. Trajectories were analysed for pose stability and fit to cryo-EM density.
Results
Functional and biochemical assays showed that ibogaine inhibits serotonin uptake with an IC50 of 5 ± 1 µM for both the ts2-active and ΔN72/C13 constructs. Addition of 5 µM ibogaine reduced Vmax by approximately 50% while Km for serotonin was unchanged, consistent with non-competitive inhibition. Saturation binding of [3H]ibogaine in NaCl to ts2-active SERT gave Kd values of 400 ± 100 nM without and 500 ± 200 nM with the 15B8 Fab. When SERT was restrained in outward-open conformations (ts2-active–15B8 Fab–8B6 scFv and ts2-inactive), [3H]ibogaine binding was weaker (Kd ≈ 5–8 µM) and competition with [3H]paroxetine yielded a Ki of 3 ± 0.4 µM, indicating roughly tenfold weaker affinity for outward-open SERT. By contrast, ibogaine bound more tightly in KCl or NMDG-Cl (Kd ≈ 130–140 nM), and 15B8 Fab did not perturb ibogaine binding in KCl (Kd ≈ 180 ± 50 nM). Electrophysiological data indicated the ibogaine-binding site is accessible from the extracellular solution. The S277C labelling experiments showed greater reactivity when SERT was bound to ibogaine than to outward-open stabilising inhibitors, particularly in KCl, consistent with increased cytoplasmic pathway accessibility upon ibogaine binding. Cryo-EM structures were determined for SERT–ibogaine complexes in three distinct conformations. A ts2-active SERT–15B8 Fab–8B6 scFv complex with ibogaine was solved in an outward-open conformation at ~4.1 Å. A ΔN72/C13 SERT–15B8 Fab–ibogaine complex in NaCl adopted an occluded conformation (~4.2 Å), and the same construct in KCl produced an inward-open conformation reconstruction at ~3.6 Å; noribogaine-bound ΔN72/C13 SERT in KCl produced a lower-resolution map (~6.3 Å) that best fitted the inward-open conformation. In all maps, density attributable to ibogaine localised to the central binding site and no alternate binding sites were observed. Docking and MD simulations, combined with map inspection, identified a consistent binding pose: the protonated tertiary amine of ibogaine forms an interaction with Asp98, while the tricyclic core lodges between aromatic residues Tyr176 and Tyr95 in outward-open and occluded states. Conserved contacts include a methoxy group extending toward a cavity between TM3 and TM8 near Asn177, Ile172 capping the tryptamine group, and Phe341 interacting with the indole region. Transition to the inward-open conformation leads to repositioning of ibogaine toward TM1a and TM8; Phe335 moves into the central site and blocks extracellular release. Mutagenesis at Asn177 supported the predicted pose: N177V exhibited substantially higher [3H]ibogaine affinity in KCl (Kd = 70 ± 20 nM, P < 0.01) and altered inhibition by ibogaine and noribogaine as expected. Comparing conformations quantified significant helical rearrangements. The extracellular gate closes during the outward-open to occluded transition primarily by movements of TM1b and TM6a (small tilts and shifts), reducing the solvent-accessible surface area of the allosteric site from 1,448 Å2 (outward-open) to 1,247 Å2 (occluded), and further to 973 Å2 in the inward-open state. The occluded-to-inward-open transition involved large changes at the intracellular gate: TM1a undergoes a hinge-like movement of ~40° into the membrane, unwinding and lateral expansion of TM5 at a GlyX9Pro motif (facilitating a ~1.8 Å intracellular shift), and TM1b tilts and shifts markedly (5.1 Å, 22°) to more fully close the extracellular vestibule. Examination of the ion sites suggested that the outward-open and occluded conformations are compatible with two bound sodium ions (Na1 and Na2), whereas inward-open rearrangements (TM5 shift and IL2 unwinding) permit Na2 access to the cytoplasm; Na1 residues also shift but may remain capable of coordinating sodium. The chloride-coordinating residues remained arranged in a manner consistent with a bound Cl- that is not directly coupled to substrate flux. Collectively, the data indicate that ibogaine is an active-site-binding inhibitor that can occupy the central site in outward-open, occluded and inward-open conformations, and that it stabilises conformations that increase cytoplasmic accessibility — a mechanistic basis for non-competitive inhibition. The authors further propose a two-step binding scenario in which ibogaine can bind from the extracellular side to outward-open SERT and then promote isomerisation to occluded or inward-open states.
Discussion
Coleman and colleagues interpret their findings to show that ibogaine binds to the central substrate site of SERT and that it can occupy and stabilise multiple transport-related conformations, including outward-open, occluded and inward-open states. They argue that this capacity to stabilise inward-open conformations explains the non-competitive character of ibogaine inhibition: serotonin does not effectively compete for binding to the inward-open state, and the ibogaine–SERT complex can exist in dynamic equilibrium with occluded states depending on ionic conditions. The structural transitions observed—particularly the hinge-like movement of TM1a, the partial unwinding and lateral expansion of TM5, and movements of TM1b and TM6a that close the extracellular gate—are placed in the context of prior models derived from bacterial NSS homologues such as LeuT. The investigators note similarities in the overarching rocker-like movement of helices against a scaffold but also highlight deviations, including heterogeneity in TM1a orientations and details of TM5 unwinding that facilitate ion and substrate release. Key limitations acknowledged by the authors include the resolution limits of some cryo-EM maps, which precluded unambiguous visualisation of ions and fine atom–atom interactions, and the use of antibody fragments and truncated or thermostabilised constructs to aid cryo-EM that could influence conformational equilibria. They also report that no statistical methods were used to predetermine sample size and that experiments were not randomised or performed blind. The authors caution that these factors should be considered when interpreting functional and conformational conclusions. In terms of implications, the study provides a structural framework for how substrate translocation and inhibitor binding are mechanistically coupled in SERT. The authors suggest that their computationally derived ibogaine poses and the demonstration that ibogaine can selectively stabilise occluded or inward-open states could inform the rational design of small molecules that target specific transporter conformations for therapeutic or research applications. They also indicate that ionic conditions, cholesterol association and specific residue substitutions can bias SERT conformational equilibria, offering routes to modulate transporter function experimentally or pharmacologically.
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THESE STRUCTURES DEFINE THE STRUCTURAL REARRANGEMENTS THAT OCCUR FROM THE OUTWARD-OPEN TO INWARD-OPEN CONFORMATIONS, AND PROVIDE INSIGHT INTO THE MECHANISM OF NEUROTRANSMITTER TRANSPORT AND IBOGAINE INHIBITION.
SERT, a monomeric membrane protein of approximately 70 kDa, poses a challenge for single-particle cryo-electron microscopy (cryo-EM); we therefore used antibody fragments to provide mass and molecular features to facilitate cryo-EM reconstruction. We also used an N-and C-terminally truncated SERT construct, denoted ΔN72/ C13, as well as three thermostable variants: ts2-active SERT, which maintains wild-type-like transport properties; ts2-inactive SERT, which is locked in the outward-open conformation; and C7x, which has no reactive cysteines. To investigate the modulation of SERT by ibogaine (Fig.), we determined the inhibition of serotonin uptake by ibogaine for the ts2-active and ΔN72/C13 SERT variants; in both cases the half-maximal inhibitory concentration (IC 50 ) was found to be 5 ± 1 µM (Extended Data Fig.). Upon the addition of 5 µM ibogaine, the maximum velocity of substrate transport (V max ) was reduced by approximately 50% and the Michaelis constant (K m ) for serotonin was unchanged (Extended Data Fig.), which is consistent with ibogaine acting as a non-competitive inhibitor. We also investigated the consequences of antibody binding, and found that the ts2-active SERT-15B8 Fab-8B6 scFv complex (Fab, antigen-binding fragment; scFv, single-chain variable fragment) is transport-inactive (Fig.) whereas the ΔN72/C13 SERT-15B8 Fab complex is transportcompetent (Fig.). Saturation binding experiments of [ 3 H]ibogaine in NaCl to ts2-active SERT without and with the 15B8 Fab yielded dissociation constants (K d ) of 400 ± 100 nM and 500 ± 200 nM (Fig.), respectively. To investigate whether ibogaine can also bind to the outward-open conformation, we carried out binding experiments on two variants in the outward-open conformation. From direct binding experiments of [ 3 H] ibogaine to the ts2-active SERT-15B8 Fab-8B6 scFv complex and the ts2-inactive variant we estimated a K d of 5-8 µM, whereas in [ 3 H]paroxetine competition experiments with the ts2-active SERT-15B8 Fab-8B6 scFv complex we measured an inhibitory constant (K i ) of 3 ± 0.4 µM (Extended Data Fig.). Together, these experiments demonstrate that the binding of ibogaine is approximately tenfold weaker when SERT is restrained in the outward-open conformation. Moreover, electrophysiological recordings show that the ibogaine-binding site is accessible from the extracellular solution, reinforcing the notion that ibogaine can bind to the transporter in the outward-open conformation. We next explored ion dependence, and found that ibogaine binds to SERT more tightly in the presence of KCl (K d = 130 ± 30 nM) or N-methyl-d-glucamine hydrochloride (NMDG-Cl; K d = 140 ± 20 nM) than in NaCl-containing buffers, in agreement with previous studies, and that the 15B8 Fab does not perturb ibogaine binding in KCl solutions (K d = 180 ± 50 nM) (Fig.). To investigate the conformation of SERT used in these studies, we examined a mutant containing a serine-to-cysteine substitution at residue 277 (S277C). This residue is located in the intracellular portion of TM5, which is solvent-accessible in the inward-open conformation(Extended Data Fig.). It has previously been reportedthat the S277C mutant in the C7x background is more reactive to methanethiosulfonate reagents when it is bound to ibogaine than when it is bound to inhibitors that stabilize the outward-open conformation (Extended Data Fig.); this differential reactivity is further pronounced in KCl in comparison to NaCl (Fig.). Together, these observations are consistent with the notion that ibogaine increases the accessibility of the cytoplasmic-permeation pathway. To locate the ibogaine-binding site of SERT and to understand how ibogaine binding influences the conformation of the transporter, we studied SERT using single-particle cryo-EM. To determine whether such studies were feasible, we carried out a 'control' reconstruction using the ts2-inactive SERT-15B8 Fab-8B6 scFv complex and the selective serotonin reuptake inhibitor, paroxetine. We discovered that the cryo-EM map is well fitted by the X-ray structure of the ts2-inactive, outward-open paroxetine complex (Extended Data Table), and that the map has clear density features for aromatic side chains and for paroxetine in the central binding site, thus demonstrating the feasibility of Letter reSeArCH single-particle cryo-EM of SERT-antibody complexes (Extended Data Fig., Extended Data Fig.). We then used the ts2-active SERT-15B8 Fab-8B6 scFv complex to investigate the binding of ibogaine to the outward-open conformation. This ibogaine-bound complex was determined at a resolution of around 4.1 Å (Fig., Extended Data Table). The TM densities were well-defined, continuous and of sufficient strength and connectivity to fit the main chain and to position many of the side chains (Extended Data Fig.). Comparison of this complex with known structures of SERT and other transporters enabled unambiguous assignment as the outward-open conformation (Extended Data Fig.-i, Extended Data Table). To further explore the conformations of SERT-ibogaine complexes, we used Fabs that preserve serotonin-uptake activity. We first elucidated the structure of the ΔN72/C13 SERT-15B8 Fab complex in NaCl, obtaining a reconstruction at a resolution of about 4.2 Å, and found that its conformation was distinct from the outward-open conformation (Extended Data Fig., Extended Data Table). Adjacent to TM1a, a density feature was found that is fit well by a molecule of cholesteryl hemisuccinate (CHS), similar to that observed in the dopamine transporter(Fig.). Comparison of the positions of TM1, TM6 and the extracellular gate to the equivalent elements of the outward-open complex indicates that, in NaCl, this SERT-15B8 Fab-ibogaine complex adopts an occluded conformation (Fig., Extended Data Fig.). In the presence of NaCl, the accessibility of residues that are located in the cytoplasmic-permeation pathway is reduced. These conditions populate the inward-closed conformation, whereas the removal of sodium increases Thr276 phosphorylationand favours the inwardopen conformation (Fig.). Thus, we examined the conformation of the ΔN72/C13 SERT-15B8 Fab-ibogaine complex in KCl; the resulting reconstruction yielded a density map at ~3.6 Å resolution (Fig., Extended Data Fig., Extended Data Table, Supplementary Video 1). At the cytoplasmic side of SERT we observed a distinct density feature associated with TM1a; this corresponds to a 'splayed' conformation of TM1a away from the transporter core, thus opening a pathway from the central binding site to the intracellular solution. The density feature for CHS near TM1a in the occluded conformation (Extended Data Fig.) was not observed in the inward conformation, which suggests that its association may be conformation-dependent. Removal of cholesterol from membranes increases the extent of ibogaine binding, and the mutation of residues that line the CHS-binding site favours the inward conformation. Non-proteinaceous features were also found near Thr276 and Ser277, sites of phosphorylation that modulate transporter conformational equilibria(Extended Data Fig.). To further explore the influence of small molecules on the conformation of SERT, we examined noribogaine, which is an ibogaine metaboliteand a non-competitive inhibitor of serotonin uptake (Fig., Extended Data Fig.). A three-dimensional (3D) reconstruction of the ΔN72/C13 SERT-15B8 Fab complex with noribogaine in 100 mM KCl yielded a density map at a resolution of 6.3 Å, enabling the visualization of helical segments (Extended Data Table). Subsequent rigid-body fitting of outward-open, occluded or inward-open conformations into the density map showed that the best fit was obtained with the inward-open conformation (Extended Data Fig.); this demonstrates that in KCl, ibogaine and noribogaine populate the inward-open conformation. The quality of the density maps enabled the localization of ibogaine at the central site, and there were no other density features attributable to ibogaine. Because the density maps are between 3.6 Å and 4.2 Å in resolution, we used computational docking followed by molecular dynamics simulations to determine the optimal binding poses of ibogaine in the central site (Fig., Extended Data Fig., Supplementary Video 2). Subsequently, we discovered that the tertiary amine of ibogaine interacts with Asp98 (Fig., b, Extended Data Fig.) while the tricyclic ring system lodges between the aromatic groups of Tyr176 and Tyr95 in the outward-open and occluded conformations. SERTibogaine interactions that are largely preserved in all three conformations include the methoxy group of ibogaine, which protrudes into a cavity between TM3 and TM8, near Asn177; Ile172, which sits 'above' the tryptamine group of ibogaine and 'restrains' the drug within the central site; and the aromatic ring of Phe341, which interacts with the adjacent indole nitrogen of ibogaine. Phe335 undergoes conformational changes in going from the outward-open and occluded to the inwardopen conformation, moving further into the central site and ultimately blocking the release of ibogaine from the extracellular side (Fig., Supplementary Video 3); meanwhile, the movement of TM1a in the inward-open conformation disrupts the interactions of Tyr95 and Asp98 with ibogaine (Fig.). Thus, upon transition of the transporter from the outward-open to the inward-open conformation, the position of ibogaine is adjusted; it moves in the direction of TM1a and TM8, towards the cytoplasmic-permeation pathway (Fig., Supplementary Video 3). When we assessed the binding pose of ibogaine, we observed that the side chain of Asn177 resides near the methoxy group of ibogaine. We reasoned that, if the pose is accurate, mutation of the asparagine to a smaller, less polar residue should enhance and diminish the affinity for ibogaine and noribogaine, respectively. We thus measured the inhibition of transport by ibogaine and noribogaine as well as the binding of [ 3 H]ibogaine for the N177V, N177A, N177T and N177L mutants, and found a more robust inhibition of 5-hydroxytryptamine (5-HT) uptake by ibogaine and a weakening of inhibition by noribogaine (Fig., Extended Data Fig.). Notably, the N177V variant has a substantially higher binding affinity than ts2-active SERT for [ 3 H] ibogaine (K d = 70 ± 20 nM, P < 0.01, one-sided Student's t-test) in KCl, which provides additional support for the binding pose of ibogaine.
LETTER RESEARCH
To further define the conformation of SERT, we next analysed the position of the extracellular and intracellular gates. We found that the ibogaine-bound, outward-open reconstruction is similar to the X-ray structure of paroxetine-bound SERT(Extended Data Fig.). Ibogaine and ions can access the central binding site from the extracellular side, because gating residues (Arg104 and Glu493, Cα-Cα distance: 12.0 Å; Tyr176 and Phe335, Cα-Cα: 13.6 Å) conform to an open gate (Fig.), while the closed intracellular gate prevents exposure to the cytoplasm. Upon formation of the occluded conformation, the core TMs of SERT undergo movements that close the extracellular gate, preventing access to the central binding site. In addition to the closure of the extracellular gate, the most substantial structural changes that occur during the transition from the outward-open to the occluded conformation are found in TMs 1b, 5, 6, 7 and 10, and in extracellular loop (EL) 6 (Extended Data Fig.). The changes associated with TM6a include a tilting of 3° and a shift of 1.9 Å towards the scaffold. EL6 also moves by 1.3 Å towards TM1b and TM6a, while TM10 tilts by 2° and shifts by 0.9 Å in the same direction. TM7 shifts by 1.3 Å in the extracellular side towards the scaffold domain, while TM5 experiences a 3.2° rotation and a 1.3 Å shift towards TM7 and TM1b (Extended Data Fig.). In NSSs, an allosteric site formed by residues in the extracellular vestibule modulates dissociation from the central site. The closure of the extracellular gate (Arg104 and Glu493: 9.7 Å; Tyr176 and Phe335: 13.5 Å) changes the nature of the extracellular vestibule: the movement of TM6a towards the scaffold results in the collapse of the allosteric site-as evidenced by a reduction in the solvent-accessible surface area (1,448 Å 2 compared with 1,247 Å 2 )-thus reducing the likelihood of association of ibogaine or similar small molecules with the allosteric site (Fig.). EL3, which connects TM5 to TM6, further packs against the extracellular halves of these TMs. Subtle changes are also observed in EL4-which experiences a minor shift towards the scaffold-and localized changes in the intracellular portion of TM5 are observed, which may facilitate the transition to the inward-open conformation and the opening of TM1a (Fig.). We also investigated the conformational transitions from the occluded to the inward-open states, finding that the most noteworthy structural rearrangements are at the closed extracellular and open intracellular gates. TM1b shifts and tilts by 5.1 Å and 22°, while TM6a moves by 3.4 Å and 5° towards the scaffold, closing the extracellular gate-as evidenced by a further reduction in the solvent-accessible surface area of the allosteric site (973 Å 2 ) and the distance between extracellular gating residues (Arg104 and Glu493: 9.9 Å; Tyr176 and Phe335: 11.0 Å) (Fig., i, Extended Data Fig.). TM2 and TM7 undergo an associated movement of 2.8 Å and 1.0 Å in the extracellular Cα marker positions, with an overall angular change of 7.3° and 4.8° towards the scaffold, respectively. A hinge-like movement of TM1a by 40° into the plane of the membrane disrupts interactions between the N terminus and the cytoplasmic half of TM6 (Tyr350, Trp82) that are present in the outward-open and occluded conformations (Cα-Cα distance: 7.0 Å in occluded); this movement opens the cytoplasmic-permeation pathway and grants accessibility to the central binding site (Fig., Extended Data Fig.). The movement of TM1a is accompanied by structural changes in TM5, which unwinds at the GlyX 9 Pro motifand expands laterally into the membrane, facilitating a shift of 1.8 Å in the intracellular side and 1 Å in the extracellular side, and an angular change of 7° (Fig., Extended Data Fig.). The net result of these movements is a constriction of the extracellular surface and an expansion of the intracellular diameter of the transporter as it transitions from an occluded to an inward-open conformation (Extended Data Fig., Supplementary Video 4). The movement of key helices against the scaffold domain mirrors the conformational changes that are observed in bacterial amino acid transporters, although deviations from the prototypical model are also present (Extended Data Fig.). The occluded conformation of SERT most closely resembles the outward-facing occluded conformation of LeuT). Given the heterogeneity observed for TM1a in the molecular dynamics simulations of LeuT 10 , it is possible that TM1a in SERT may also sample different orientations upon the rupture of the intracellular gate. To gain insight into the occupancy of the sodium sites in the outward-open, occluded and inward-open conformations, we examined the conformations of the surrounding protein residues because, at the current resolutions, we were unable to resolve density for ions. In the outward-open and occluded conformations, the positions of residues surrounding Na1 and Na2 sites suggest that these conformations are compatible with two bound sodium ions (Extended Data Fig.). The shift of TM5 towards the membrane, together with the unwinding of intracellular loop (IL) 2, enables Na2 to access the cytoplasm in the inward-open conformation (Fig., Extended Data Figs.); this is similar to MhsT, in which the unwinding of TM5 at the GX 9 P motif is also thought to result in the release of sodium from the Na2 site. Sodium-coordinating residues at the Na1 site also undergo considerable displacement in the inward-open conformation, although their arrangement suggests that they may still be capable of ion binding. The arrangement of chloride-coordinating residues is also consistent with an occupied Cl -site that is not coupled to substrate flux. Thus, the positions of the ion sites suggest distinct roles of Na1, Na2, and Cl -, in which Na2 may be directly coupled to substrate transport. We observe that ibogaine interacts with SERT in outward-open, occluded and inward-open conformations, and can be classified as an active-site-binding inhibitor that displays non-competitive inhibition characteristics. Because ibogaine cannot directly access the central binding site in the occluded conformation, we speculate that ibogaine binds to either the outward-open or inward-open conformation, suggesting the possibility that it may remain bound and enable transporter isomerization (Fig.). Binding of ibogaine to the inward conformation probably forms the basis for the non-competitive inhibition of transport, because serotonin does not compete for binding to this conformation and the SERT-ibogaine complex may exist in dynamic equilibrium with the occluded conformation, depending on the ionic conditions.
LETTER RESEARCH
Ibogaine binding in KCl is consistent with an isomechanistic mechanismvia direct binding to the inward-open conformation. However, the observation of an extracellularly accessible ibogaine-binding siteis suggestive of a more complex two-step mechanism, in which the first step involves binding to an outward-open conformation and the second step involves the stabilization of an occluded or inward-open conformation. The larger steric bulk of ibogaine compared to serotonin may preclude it from binding and unbinding from the central site through the intracellular pathway without considerable conformational fluctuation, even in the inward-open conformation, thus explaining why ibogaine is not a substrate (Extended Data Fig.). We nevertheless exploited the observation that ibogaine stabilizes multiple conformations of the transporter, in conjunction with specific ions or arresting antibody fragments, using its complexes with SERT to provide insight into how the transporter isomerizes from outward-open to occluded and inward-open conformations. Moreover, our computational determination of the pose of ibogaine in the occluded and inward-open states provides fresh insight into how high-affinity small molecules might be crafted to selectively bind to the occluded or inward-open conformations of SERT.
DATA REPORTING.
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Antibody production. The 15B8 Fab was produced either by papain digestion of 15B8 mAb and purification by cation-exchange chromatography, using standard methods, or by isolation of recombinantly expressed Fab from Sf9 supernatant by metal affinity chromatography for crystallization. The 8B6 heavy and light chains of the variable domain were fused to a PelB signal sequence, an N-terminal 8-His tag, and connected by a (GGGGS) 3 linker to create the 8B6 scFv. The 8B6 scFv was expressed overnight at 25 °C in BL21 cells induced with 0.1 mM isopropyl-β-d-1-thiogalactopyranoside. Periplasmic proteins were extracted by homogenizing cells in 200 mM Tris pH 8, 20% sucrose, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. The buffer was exchanged to 50 mM phosphate pH 8.0, 300 mM NaCl and 10 mM imidazole by dialysis. The 8B6 scFv was purified by metal affinity chromatography and size-exclusion chromatography on a Superdex 75 column. SERT expression and purification. The human SERT constructs used in this study are the N-and C-terminally truncated wild type (ΔN72, ΔC13), ts2-active (I291A, T439S), C7x made in the ts2-active background (C109A, C147A, C155S, C166L, C522S, C357L, C369L), and ts2-inactive (Y110A, I291A). The expression and purification of the aforementioned constructs was carried out as described previouslywith minor changes. SERT was expressed as a C-terminal GFP fusion using baculovirus-mediated transduction of HEK-293S GnTI -cells (ATCC). HEK-293S GnTI -cells were not authenticated and all cell lines tested negative for mycoplasma contamination. Cells were solubilized in 20 mM Tris pH 8 with 150 mM NaCl or 100 mM KCl, containing 20 mM n-dodecyl-β-d-maltoside (DDM) and 2.5 mM CHS, in the presence of 1 µM paroxetine, or 10 µM ibogaine, or 10 µM noribogaine, and were then purified into 1 mM DDM, 0.2 mM CHS, and 1 µM paroxetine, or 10 µM ibogaine, or 10 µM noribogaine in 20 mM Tris pH 8 with 100 mM NaCl or 100 mM KCl by Strep-Tactin affinity chromatography. The Nand C-terminal GFP and purification tags were removed by thrombin digestion. For the SERT-Fab-scFv complex, 15B8 Fab and 8B6 ScFv were mixed with SERT at a 1:1.2:1.2 molar ratio and purified by size-exclusion chromatography on a Superdex 200 column in TBS (20 mM Tris pH 8, 100 mM NaCl) containing 9 mM nonylmaltoside, 0.2 mM CHS, and 1 µM paroxetine or 10 µM ibogaine. For the ΔN72/C13 SERT-Fab complex, 15B8 Fab was mixed with SERT at a 1:1.2 ratio and separated by size exclusion on a Superdex 200 column in 20 mM Tris pH 8, 100 mM NaCl or 100 mM KCl containing 1 mM DDM, 0.2 mM CHS, and 10 µM ibogaine or 10 µM noribogaine. The peak fraction containing the SERT complexes was concentrated to 4 mg ml -1 before the addition of 250 µM paroxetine, 1 mM ibogaine or 1 mM noribogaine. Crystallization of 15B8 Fab. The 15B8 Fab was crystallized by hanging-drop vapour diffusion (Extended Data Table). Crystals appeared after several days using a reservoir solution composed of 80 mM sodium citrate pH 5.2 and 2.2 M ammonium sulfate at a 1:1 ratio of protein:reservoir. The 15B8 Fab crystals were cryoprotected with 25% ethylene glycol before flash-cooling in liquid nitrogen. Cryo-EM sample preparation and data acquisition. The SERT-antibody complexes (2.5 µl), at a concentration of 40-80 µM, were applied to glow-discharged Quantifoil holey carbon grids (gold, 1.2/1.3 or 2.0/2.0 µm size/hole space, 200 mesh). For 'multi-shot' data collection, 100 µM fluorinated n-octyl-β-d-maltoside (final concentration) was added to the sample before freezing. The grids were blotted for 1.5-2.5 s at 100% humidity using a Vitrobot Mark IV, followed by plunging into liquid ethane cooled by liquid nitrogen. Images were acquired using a FEI Titan Krios equipped with a Gatan Image Filter operating at 300 kV or an Arctica transmission electron microscope (TEM) at 200 kV. A Gatan K2 Summit direct electron detector was used, on both TEMs, to record movies in super-resolution counting mode with a binned pixel size of 1.044 or 0.823 Å per pixel on the Krios or 0.910 Å per pixel on the Arctica, respectively. The typical defocus values ranged from -1.0 to -2.5 µm. Exposures of 8-10 s were dose-fractionated into 40-100 frames, resulting in a total dose of 50-60 e -Å -2 . Images were recorded using the automated acquisition program SerialEM. Image processing. Micrographs were corrected for beam-induced drift using MotionCor2. The contrast transfer function (CTF) parameters for each micrograph were determined using Gctf. Particles were picked using DoG-Picker. Particles were subjected to reference-free 2D classification in either RELION 2.1or cryoSPARCfollowed by homogenous refinement in cryoSPARC. Local refinement was performed in cisTEMwith a mask which excludes the micelle and Fab constant domain to remove low-resolution features (Extended Data Figs.). The molar masses of the SERT-15B8 Fab-8B6 scFv and SERT-15B8 Fab complexes were 135 and 105 kDa respectively. Focused 3D classificationwas also performed in cisTEM using a spherical mask centred on SERT to discover additional conformational heterogeneity. The resolution of the reconstructions was assessed using the Fourier shell correlation (FSC) criterion and a thresholdof 0.143 in cisTEM. The low-resolution refinement limit was incrementally increased while maintaining a correlation of 0.95 or greater until no further improvement in map quality was observed. The FSC of the model versus the full map and half maps was calculated using the standalone program calculate_fsc, which is part of the cisTEM package. The local resolution was calculated using RELION 3.0. Maps were sharpened using cisTEM unless otherwise noted. For the ts2-inactive paroxetine Fab-scFv dataset, a total of 1,278,876 particles with a box size of 240 square pixels was selected from 2,904 micrographs (Extended Data Fig.). After two rounds of 2D classification using cryoSPARC, particles that had clearly defined and recognizable features were combined for further analysis. CryoSPARC was used to generate an ab initio model with two classes. Particles belonging to a class with well-defined features were further refined using local refinement in cisTEM. The low-resolution limit cut-off for refinement was 7.5 Å. The map was sharpened using local sharpening in PHENIX. For the ts2-active ibogaine Fab-scFv dataset, a total of 592,117 particles with a box size of 300 pixels was selected from 1,639 micrographs. After multiple rounds of 2D classification and ab initio reconstruction using cryoSPARC, 153,986 particles that had clearly defined features were selected. Particle coordinates were used to calculate the local CTF using Gctf and local refinement was performed in cisTEM (Extended Data Fig.). The low-resolution limit cut-off for refinement was 7.5 Å. The optimal sharpening B factor of -400 Å 2 inside the same mask used for refinement was determined by comparing map features for various sharpening factors in cisTEM. A similar strategy was used for the ΔN72/C13-15B8 Fab complex with ibogaine in NaCl. A total of 2,615,403 particles with a box size of 360 pixels were selected from 10,632 micrographs followed by rounds of 2D classification, ab initio reconstruction and homogeneous refinement using cryoSPARC. The final particle set contained 724,394 particles, which were subjected to local refinement using cisTEM (Extended Data Fig.). The low-resolution limit cut-off for refinement was 7.0 Å. For the ΔN72/C13-15B8 Fab complex with ibogaine in 100 mM KCl containing buffer, 1,220,861 particles with a box size of 380 pixels were selected from 7,732 micrographs. After multiple rounds of 2D classification, ab initio reconstruction, 3D classification in RELION 2.1 and homogeneous refinement using cryoSPARC, 383,617 particles were subjected to local refinement in cisTEM. (Extended Data Fig.). The low-resolution limit cut-off for refinement was 7.5 Å. Model building and refinement. Interpretation of the cryo-EM maps exploited rigid-body fitting of the higher-resolution SERT and antibody models derived from X-ray crystallography. Although the quality of the EM maps precludes a precise analysis of atom-atom interactions, we were able to model the main chain of SERT and position most of the bulky side chains. A starting model was generated by fitting SERT (Protein Data Bank (PDB) code: 6AWN)into the outward-open ibogaine-bound reconstruction together with the variable domains of 8B6 (PDB code: 5I66) and 15B8 (PDB code: 6D9G, Extended Data Table) Fabs derived from high-resolution crystal structures in Chimera. Model refinement was performed in Rosetta using iterative local rebuilding. Models were scored according to the fit to the density and overall Rosetta score. The best models were selected and used as templates for further refinement in RosettaCM. The paroxetine-bound model was refined separately in RosettaCM starting from SERT (PDB code: 6AWN) with the variable domains of 8B6 and 15B8. To build the occluded and inward-open conformation models, the 8B6 variable domain was removed from the outward-open ibogaine ts2-active model, followed by fitting of the model into the occluded or inward-open reconstructions. Several rounds of iterative local rebuilding were performed, followed by combining pieces from multiple templates and refinement with RosettaCM. The final stages of model building involved manual adjustments and building where merited by the quality of the electron microscopy maps in Coot, followed by real space refinement in PHENIX. For cross-validation, the FSC curve between the refined model and half maps was calculated and compared to avoid overfitting. MolProbity was used to evaluate the stereochemistry and geometry of the structures. For the outward-open and occluded reconstructions, SERT residues 79-615 were modelled into the cryo-EM maps, whereas residues 78-617 were modelled for the inward-open reconstruction. This strategy, coupled with docking and molecular dynamics simulations, furthered our interpretation of the large-scale rearrangements of structural elements in each conformation and provided a basis for molecular details of ibogaine interactions within the central site. Figures were prepared in PyMOL 51 and Chimera. The profile of the intracellular pathways shown in Extended Data Fig., c was calculated using CAVER. Measurements. All distance measurements were calculated from Cα positions. Extracellular measurements were made from Tyr186 in TM3 to marker positions in TM1b (Gln111), TM2 (Ala116), TM4 (Gln254), TM5 (Gly299), TM6a (Asp328), TM7 (Met386), TM8 (Thr421), TM9 (Thr480), TM10 (Ala486), TM11 (Phe556) and TM12 (Ser574). Intracellular measurements were made from Gly160 in TM3 to marker positions in TM1a (Lys85), TM2 (His143), TM4 (Tyr267), TM5 (Trp282), TM6b (Ser349), TM7 (Tyr358), TM8 (Glu453), TM9 (Arg462), TM10 (Phe515), TM11 (Trp535) and TM12 (Ile599). To measure the angular change between conformations, the TM helices were superimposed and the angle between helices was measured using Cα positions in PyMOL. The uncertainty of each measurement and the position of ibogaine was calculated from 100 models which were randomly perturbed by ~1.0 Å r.m.s.d. and real space refined 'back' into each map in PHENIX, as described. The solvent-accessible surface area of the allosteric site was calculated from residues within 5 Å of the (S)-citalopram structure (PDB code: 5I73; residues 100, 103-105, 175, 327-338, 368, 490-503, 549-557, 561, 563, 579).
PROTEIN PREPARATION FOR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS.
The outward-open ts2-active, occluded ΔN72/C13, and inward-open ΔN72/C13 conformations of SERT were prepared for simulations by removing antibody fragments, adding missing hydrogen atoms and side chains in the psfgen (. ks.uiuc.edu/Research/vmd/plugins/psfgen/)plugin of VMD, and by removing CHS from the occluded conformation. Because we worked with a penultimate version of experimental coordinates for the outward-open conformation, residues Gly83, Lys84, Thr219 and Trp220 had cis peptide bonds, which we 'flipped' to a trans conformation using the Cispeptide 55 plugin of VMD. We note that the deposited experimental outward-open structure has trans peptide conformations at these residues. Glu136 and Glu508 were modelled with protonated side chains according to pK a calculations using PROPKA 3.0for the outward-open and inward-open conformations. For the outward-open and occluded conformations, two Na + ions and one Cl -ion were modelled on the basis of the (S)-citalopram and paroxetine-bound X-ray structures of SERT (PDB codes: 5I71 and 5I6X), while a Cl -ion was modelled in the inward-open conformation. The models were aligned with the orientation of the paroxetine-bound SERT crystal structure (PDF code: 5I6X) from the OPM (Orientation of Protein in Membranes) database, available at. Force field parameterization. The force field parameters of protonated ibogaine were developed on the basis of the CHARMM General Force Field (CGenFF). The atom types and initial parameters were determined using the CGenFF webserver (), and the parameters were further optimized using the Force Field Toolkit (ffTK)plugin of VMD. The detailed strategy for the optimization of parameters was as follows. First, partial atomic charges were assigned to aliphatic carbon and hydrogen atoms according to the convention of CHARMM force fields (+0.09e per aliphatic hydrogen, neutralized by the negative charge assigned to the carbon atom carrying hydrogens). The partial atomic charges of the methoxyindole group were optimized according to the calculated water interactions of the corresponding atoms in the model compound (5-methoxy-2, 3-dimethylindole) at the HF/6-31G* level of theory. The partial charges of the tertiary amine group were assigned as protons at +0.32e and nitrogen at -0.40e, and all three α-carbons were assigned equal partial charges at +0.21e so that a sum of +1e net charge at the tertiary amine group was satisfied. This partial charge assignment scheme is based on those of other tertiary amine species in CGenFF (for example, N-methylpiperidine). The bonded parameters of ibogaine also contain 2 novel bonds, 11 novel angles, and 41 novel dihedrals that were not defined in the standard CGenFF force field. All of these parameters except two dihedrals were directly adopted from the initial parameter set generated from the CGenFF webserver through analogy to existing parameters without further optimization, a standard procedure for CGenFF parameters with low predicted penalty scores(assigned by the CGenFF webserver). The only two dihedral terms that were optimized were both centred around the rotation of the bond connecting positions 2 and 3 of the indole ring, which were calibrated with a 180° dihedral scan at 15° intervals using the MP2/6-31G* level of theory on the model compound 5-methoxy-2,3-dimethylindole. All quantum mechanical calculations were performed using Gaussian 09 (ref.) (). Computational search for docking poses of ibogaine. A workflow (Extended Data Fig., Supplementary Video 2) was developed to systematically search for optimal binding poses of ibogaine in the outward-open, occluded, and inwardopen conformations of SERT, independently. Using the approximate geometric centre of the binding pocket (defined as Tyr95, Ala96, Asp98, Ile172, Ala173, Tyr175, Phe335, Ser336, Gly338, Phe341, Ser438, Gly442, Leu443, Thr497, Gly498 and Val501) as the origin, a 6 × 6 × 6 search grid (1 Å spacing) was defined. For each conformation of SERT, an energy-minimized copy of ibogaine was placed at every grid point, rotated around all combinations of three Euler angles (at 18° intervals) and all six rotameric forms of the methoxy and ethyl groups, which resulted in 7.5 million SERT-ibogaine models with different ibogaine poses. These poses were analysed using the following four steps: first, a 20-step energy minimization of all the SERT-ibogaine models in NAMD2to remove straightforward steric clashes, during which the protein backbone was not allowed to move; second, calculating pair interaction energy (PIE) between ibogaine and the protein by evaluating the sum of van der Waals and electrostatic interaction energies with the pair interaction module in NAMD2, only ibogaine poses with negative (favourable) PIE are included in the clustering step; third, clustering of binding poses of ibogaine on the basis of the mass-weighted r.m.s.d. of ibogaine using a hybrid k-centres k-medoids clustering methodwith a 2-Å cut-off; and fourth, disposing insignificant clusters of binding poses, defined as those with <1% population, or those with negative ΔCCC (the difference in the cross-correlation coefficient with the cryo-EM density between the model with and the model without ibogaine). Steps 1-4 were iterated until the number of clusters converged. From the resulting final clusters (20 clusters for outward-open, 30 for occluded and 46 for inward-open), the SERT-ibogaine model with the strongest PIE in each cluster was selected for an additional 3,000 steps of minimization and a 10-ns molecular dynamics simulation in NAMD2(96 independent simulations in total) in a membrane environment (see Methods section 'Molecular dynamics simulations'). During these steps the protein backbone atoms were harmonically restrained with a 1 kcal mol -1 Å -2 force constant. Simulation trajectories were recorded every 10 ps. The first 2 ns were discarded to allow for equilibration, resulting in 800 snapshots of ibogaine per simulation. For each SERT conformation, the resulting snapshot sets (20 for outward-open, 30 for occluded and 46 for inward-open; each set with 800 snapshots) were ranked by their averaged ΔCCC, from which the best snapshot set was selected for each SERT conformation. From these highest-ranked sets, the ibogaine pose with the highest ΔCCC was selected as the optimal pose for each conformation. Molecular dynamics simulations. The following procedures were used for all the molecular dynamics simulations performed on ligand-bound SERT systems. Each SERT-ibogaine model was first internally hydrated by adding water molecules with the Dowser 65 plugin of VMD, followed by the insertion of the hydrated protein into a lipid bilayer composed of 236 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules obtained from CHARMM-GUI, and solvated with NaCl (~100 mM, for outward-open and occluded) or KCl (~100 mM, for inward-open) in VMD, resulting in a box with approximate dimensions of 100 × 100 × 105 Å 3 that contained around 100,000 atoms. To investigate the stability of the ibogaine binding poses determined by the ligand-docking procedure, two 50-ns simulations were performed with each ibogaine-bound conformation (three systems in total) in a POPC lipid bilayer, resulting in six trajectories. After 3,000 steps of minimization, the systems were equilibrated for 600 ps, during which Cα atoms, non-hydrogen atoms of ibogaine and the bound ions were restrained by harmonic potentials with decreasing force constants (k = 1, 0.5, and 0.1 kcal mol -1 Å -2 for 200 ps each) to allow for relaxation of protein side chains and hydration of the protein. Weak harmonic potentials (k = 0.1 kcal mol -1 Å -2 ) were applied to the Cα atoms (excluding N-and C termini). The same simulation protocols were applied to both 10-ns and 50-ns molecular dynamics simulations. All simulations were performed using NAMD2and CHARMM36m force fieldsfor SERT, CHARMM36 force fieldsfor lipids, and the TIP3P modelfor water. The force field parameters for ibogaine were developed on the basis of the CGenFF 58 , with further optimization using ffTK (see Methods section 'Force field parameterization'). All simulations were carried out as isothermal-isobaric (NPT) ensembles, in which the system pressure was independently coupled along the xy (membrane plane) and z (membrane normal) dimensions to allow for their independent changes. A constant temperature of 310 K was maintained using Langevin dynamics with a 1.0-ps -1 damping coefficient, and a constant pressure of 1.01325 bar was maintained with the Langevin piston Nosé-Hoover method. Non-bonded interactions were calculated in a pairwise manner within the 12-Å cut-off, with a switching function applied between 10 Å and 12 Å. Long-range non-bonded interactions were calculated with the particle mesh Ewald (PME) method. Bond lengths involving hydrogen atoms were fixed using the SHAKE algorithm. Simulations were integrated in 2-fs time steps, and trajectories recorded every 10 ps. Data analysis of simulation trajectories. Hybrid k-centres k-medoids clusteringwas performed with MDToolbox ().Trajectory analysis was carried out in VMDand MDAnalysis. VMDwas used for visualization. The cross-correlation coefficient between the cryo-EM map and the model was calculated using the Molecular Dynamics Flexible Fitting plugin. The PIE was calculated in NAMD2. The r.m.s.d. and distance plots were smoothed using a sliding window of 50 frames (0.5 ns). Reconstitution and labelling of SERT in nanodiscs. The S277C mutant was introduced into the C7x variant, in which the most reactive endogenous cysteines have been mutated to non-reactive residues. For labelling studies, purified SERT was mixed with soybean asolectin and MSP1E3D1 (ref.) and reconstituted into nanodiscs at a molar ratio of 1:5:400. Detergent was removed by incubation with Bio-Beads overnight at room temperature, followed by size-exclusion chromatography on a Superdex 200 column in TBS. SERT in nanodiscs was incubated with 1 mM ibogaine or 0.2 mM paroxetine for 30 min at room temperature, followed by labelling with 10 µM MTS-ACMA for the indicated time. Labelled SERT was desalted and analysed on a non-reducing SDS-PAGE gel. Radioligand binding and uptake assays. To measure uptake, 1 × 10 5 HEK-293S GnTI -cells transduced with ts2-active, Asn177 mutants or ΔN72/C13 SERT were plated into 96-well plates coated with poly-d-lysine. After 24 h, cells were
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washed with uptake buffer (25 mM HEPES-Tris, pH 7.0, 130 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgSO 4 , 1 mM ascorbic acid and 5 mM glucose). In selected instances, antibodies were added to cells at a concentration of 1 µM, which is in excess of the estimated K D for the 8B6 scFv and the 15B8 Fab (<10 nM). To measure the background counts, 100 µM paroxetine was added to the cells. [ 3 H]5-HT diluted 1:500 with unlabelled 5-HT, or [ 14 C]5-HT at concentrations of 0.03-40.0 µM was also added to the cells. In the case of [ 3 H]5-HT, uptake was stopped by rapidly washing cells three times with 100 µl uptake buffer, solubilizing with 20 µl of 1% Triton-X100, followed by addition of 200 µl of scintillation fluid to each well. The amount of labelled 5-HT was measured by counting in a standard 96-well plate or in a Cytostar-T plate using a MicroBeta scintillation counter. Data were fit to a Michaelis-Menten equation. Competition binding experiments were performed using scintillation proximity assays (SPA) with 5 nM SERT, 0.5 mg ml -1 Cu-YSi beads in TBS containing 1 mM DDM, 0.2 mM CHS, and 5 nM [ 3 H]paroxetine and at 0.1 nM-1 mM of the cold competitors. Where indicated, antibodies were added to SERT at a concentration of 1 µM. Experiments were measured in triplicate, and each experiment was performed three times. The error bars for each data point represent the s.e.m. K i values were determined with the Cheng-Prusoff equationin GraphPad Prism. Ibogaine binding was measured via SPA using ts2-active SERT purified in SPA buffer (20 mM Tris pH 8, 100 mM NaCl or 100 mM KCl containing 50 µM lauryl maltose neopentyl glycol and 10 µM CHS). Each experiment contained SERT at a concentration of 50 nM mixed with 0.5 mg ml -1 Cu-YSi beads and [ 3 H]ibogaine at a concentration of 15-4,000 nM (1:10 hot:cold) in SPA buffer. Non-specific binding was estimated by experiments that included 100 µM unlabelled paroxetine. In the case of binding experiments in KCl or NMDG, the buffer in the non-specific binding experiments was supplemented with 25 mM NaCl to ensure the sodium-ion-dependent binding of paroxetine. Data were analysed using a single-site binding function. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this paper. The experiment was performed three times independently with the same results. b, Michaelis-Menten plots of 5-HT uptake for wild-type (blue) transporter in the absence (solid line, circles), or in the presence (dashed line, circles) of 5 µM ibogaine, and for ts2 (red) in the absence (solid line, squares), or in the presence (dashed line, squares) of 5 µM ibogaine. Data are mean ± s.e.m. (n = 3 biological replicates.). The experiment was performed three times independently with the same results. The mean K m and error (s.e.m.) of curve fitting for ΔN72/C13 is 2.2 ± 0.3 µM; and for ts2-active is 4 ± 1 µM. c, Competition binding of ibogaine with [ 3 H]paroxetine for ts2 in the absence (filled squares) or presence (open squares) of 1 µM 15B8 and 8B6 yields a K i value of 3.2 ± 0.4 µM. Data are mean ± s.e.m. of curve fitting (n = 3 technical replicates). The experiment was performed three times independently with the same results. d, [ 3 H] ibogaine saturation binding experiments of ts2-inactive and ts2-active 15B8 Fab-8B6 scFv complex in 100 mM NaCl, and corresponding mean K d values derived from the curve fit: ts2-inactive (filled squares, >5 µM), ts2-active 15B8 Fab-8B6 scFv complex (open triangles, >8 µM). Data are mean ± s.e.m. (n = 6 biological replicates). The experiment was performed twice with similar results. e, SDS-PAGE of S277C labelling with MTS-ACMA compared with the C7x construct in nanodiscs in the presence of 1 mM ibogaine and 100 mM NaCl. There is no detectable labelling of the C7x construct. The experiment was performed three times independently with similar results. f, Time-dependent labelling of S277C (background construct: ts2-active, C7x) with MTS-ACMA in the presence of ibogaine (filled circles) and paroxetine (open squares) in 100 mM NaCl. Data are mean ± s.e.m. (n = 3 technical replicates). The experiment was performed three times with similar results. g, Analysis of S277C labelling experiments using MTS-ACMA in the presence of ibogaine or paroxetine, analysed by SDS-PAGE and visualized by in-gel fluorescence. The experiment was performed three times independently with similar results. h, Three-dimensional reconstruction and fit to the density map with the model derived from the paroxetine-bound X-ray structure (PDB code: 6AWN). SERT is cyan, 15B8 is purple and 8B6 is green; TM1 and TM6 are orange and red, respectively. i, The fit of paroxetine into the electron-microscopy density map (blue mesh) and interacting residues. j, Left, details of the 15B8-SERT interface with the EL2 region shown as an electrostatic surface potential map and 15B8 shown in ribbon representation. The Fab is coloured dark blue (heavy chain) or light blue (light chain); selected Fab residues within 5 Å of SERT are shown as sticks. Right, a similar view but with the Fab shown as a semitransparent electrostatic surface potential. EL2 of SERT is shown in ribbon representation and is coloured cyan.
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Extended Data Fig.| Cryo-EM reconstruction of ts2-active SERT-15B8 Fab-8B6 scFv-paroxetine complex. a, Workflow of cryo-EM data processing of the ts2-inactive SERT-15B8 Fab-8B6 scFv complex with paroxetine in the outward-open conformation. After particle picking, particles were sorted using 2D classification. 3D ab initio reconstructions were performed after 2D classification using cryoSPARC. One out of two predominant classes (boxed) exhibited a subset of homogeneous particles that were used for further processing and global alignment in cryoSPARC. The other class, upon refinement, yielded only a nanometreresolution map. Local refinement using cisTEM improved the resolution of class 1 (boxed) upon masking of the Fab constant domain and micelle (mask is shown overlaid in blue on top of the reconstruction). The final reconstructed volume was sharpened using PHENIX. b, Representative cryo-EM micrograph. Individual single particles are circled in white. Scale bar, 50 nm. c, 2D class averages after three rounds of classification. d, The angular distribution of particles used in the final reconstruction. e, Cryo-EM density map coloured by local resolution estimation. f, FSC curves for cross-validation, the final map (blue), masked SERT-Fab complex (red), and a mask that isolated SERT (black). The low-resolution limit cut-off for refinement was 7.5 Å. g, FSC curves for model versus half map 1 (working, red), half map 2 (free, black) and model versus final map (blue). h, Cryo-EM density segments of TM1 to TM12. i, A spherical mask placed over SERT was used for focused 3D classification with 3 classes. Comparison of the classes did not reveal any substantial differences. The antibodies were removed for clarity. The number of particles belonging to each class average is: class 1, purple (11.9%, 25,530 particles); class 2, yellow (54.9%, 117,781 particles); class 3, cyan (33.2%, 71,226 particles).
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Extended Data Fig.| Cryo-EM reconstruction of ts2-active 15B8 Fab-8B6 scFv-ibogaine complex. a, Workflow of cryo-EM data processing of the ts2-active 15B8 Fab-8B6 scFv complex with ibogaine in the outward-open conformation. After particle picking, particles were sorted using 2D classification. Ab initio reconstructions were performed in cryoSPARC after 2D classification to obtain an initial reconstruction. Particles were used for further processing and global alignment in cryoSPARC followed by recentring in RELION and calculation of the local CTF using Gctf. Local refinement using cisTEM improved the resolution upon masking of the Fab constant domain and micelle (mask is shown overlaid in blue on top of the reconstruction). The final reconstructed volume was sharpened using cisTEM. b, Representative cryo-EM micrograph. Individual single particles are circled in white. Scale bar, 50 nm. c, 2D class averages after three rounds of classification. d, The angular distribution of particles used in the final reconstruction. e, Cryo-EM density map coloured by local resolution estimation. f, FSC curves for cross-validation, the final map (blue), masked SERT-Fab complex (red), and a mask that isolated SERT (black). The low-resolution limit cut-off for refinement was 7.5 Å. g, FSC curves for model versus half map 1 (working, red), half map 2 (free, black) and model versus final map (blue). h, Cryo-EM density segments of TM1 to TM12. i, A spherical mask placed over SERT was used for focused 3D classification with 3 classes. Comparison of the classes did not reveal any substantial differences. The antibodies were removed for clarity. The number of particles belonging to each class average is: class 1, purple (33.6%, 51,739 particles); class 2, yellow (38.8%, 59,747 particles); class 3, cyan (27.6%, 42,500 particles).
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Extended Data Fig.| Cryo-EM reconstruction of ΔN72/C13 SERT-15B8 Fab-ibogaine complex in NaCl. a, Workflow of cryo-EM data processing of the ΔN72/C13 SERT-15B8 Fab complex with ibogaine in NaCl in the occluded conformation. After particle picking, particles were sorted using 2D classification. Ab initio reconstructions were performed in cryoSPARC after 2D classification to obtain an initial reconstruction. Particles were used for further processing and global alignment in cryoSPARC followed by recentring in RELION and calculation of the local CTF using Gctf. Local refinement using cisTEM improved the resolution upon masking of the Fab constant domain and micelle (mask is shown overlaid in blue on top of the reconstruction). The final reconstructed volume was sharpened using cisTEM. b, Representative cryo-EM micrograph. Individual single particles are circled in white. Scale bar, 50 nm. c, 2D class averages after three rounds of classification. d, The angular distribution of particles used in the final reconstruction. e, Cryo-EM density map coloured by local resolution estimation. f, FSC curves for cross-validation, the final map (blue), masked SERT-15B8 Fab complex (red), and a mask that isolated SERT (black). The low-resolution limit cut-off for refinement was 7.0 Å. g, FSC curves for model versus half map 1 (working, red), half map 2 (free, black) and model versus final map (blue). h, Cryo-EM density segments of TM1 to TM12. i, A spherical mask placed over SERT was used for focused 3D classification with 3 classes. Comparison of the classes did not reveal any substantial differences. The Fab was removed for clarity. The number of particles belonging to each class average is: class 1, purple (78.9%, 571,547 particles); class 2, yellow (6.9%, 49,983 particles); class 3, cyan (14.2%, 102,863 particles).
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Extended Data Fig.| Cryo-EM reconstruction of ΔN72/C13 SERT-15B8 Fab-ibogaine complex in KCl. a, Workflow of cryo-EM data processing of the ΔN72/C13 SERT-15B8 Fab complex with ibogaine in KCl in the inward-open conformation. After particle picking, particles were sorted using 2D classification. Ab initio reconstructions were performed in cryoSPARC after 2D classification to obtain an initial reconstruction. Particles were further sorted in RELION using 3D classification and refined further in cryoSPARC. Local refinement using cisTEM improved the resolution upon masking of the Fab constant domain and micelle (mask is shown overlaid in blue on top of the reconstruction). The final reconstructed volume was sharpened using cisTEM. b, Representative cryo-EM micrograph. Individual single particles are circled in white. Scale bar, 50 nm. c, 2D class averages after three rounds of classification. d, The angular distribution of particles used in the final reconstruction. e, Cryo-EM density map coloured by local resolution estimation. f, FSC curves for cross-validation, the final map (blue), masked SERT-Fab complex (red), and a mask that isolated SERT (black). The low-resolution limit cut-off for refinement was 7.5 Å. g, FSC curves for model versus half map 1 (working, red), half map 2 (free, black) and model versus final map (blue). h, Cryo-EM density segments of TM1 to TM12. i, A spherical mask placed over SERT was used for focused 3D classification with 3 classes. Comparison of the classes did not reveal any substantial differences. The Fab was removed for clarity. The number of particles belonging to each class average is: class 1, purple (32.9%, 121,288 particles); class 2, yellow (33.7%, 124,237 particles); class 3, cyan (33.4%, 123,131 particles). Extended Data Fig.| Cholesteryl hemisuccinate, map features at Thr276 and Ser277, and SERT-noribogaine complex. a, Interaction between CHS, TM1a and TM5 in the occluded conformation of the ΔN72/C13 SERT-15B8-ibogaine complex in 100 mM NaCl. b, Non-proteinaceous density features (red) near Thr276 and Ser277. c, Noribogaine inhibition of 5-HT transport for ΔN72/C13 SERT. 5-HT transport was measured using 20 µM [ 3 H]5-HT in the presence of the indicated concentrations of noribogaine. The mean IC 50 of noribogaine inhibition of serotonin transport was determined from the curve with the error of the fit (s.e.m.): 1.2 ± 0.2 µM. Data are mean ± s.e.m. (n = 3 biological replicates). The experiment was performed twice independently with similar results. d, Michaelis-Menten plots of 5-HT uptake for the ΔN72/C13 transporter in the absence (solid line, circles), or in the presence (dashed line, squares) of 1 µM noribogaine; the mean K m was determined from the curve with the error of the fit (s.e.m.): ΔN72/C13: 2.7 ± 0.6 µM; in the presence of noribogiane: 2.7 ± 0.9 µM. Data are mean ± s.e.m. (n = 3 biological replicates). e, Noribogaine (solid line, circles) and ibogaine (dashed line, squares) competition binding with [ 3 H] paroxetine for ΔN72/C13 SERT. Data are mean ± s.e.m. (n = 3 technical replicates). f, Density map of the ΔN72/C13 SERT-15B8-noribogaine complex, in 100 mM KCl, fit with the model derived from the inward-open ibogaine-bound SERT complex. SERT is cyan and the 15B8 Fab is purple; TM1 and TM6 of SERT are shown in orange and red, respectively. g, Noribogaine density in the central binding pocket. The fit of noribogaine into the electron microscopy density map was derived from ibogainebound SERT in the inward-open conformation and is shown in blue mesh, and residues involved in binding (Tyr176, Asp98, Phe341, Phe335, Asn177, Ile172 and Tyr95) are drawn as sticks. h, FSC curve for the noribogainebound SERT complex. The low-resolution limit cut-off for refinement was 9.0 Å.