Structural mechanism of substrate binding and inhibition of human taurine transporter

Domain: chemistry
Published: 2026-03-20
Editorial outcome: major_revision

Abstract
Taurine is a sulfur-containing amino acid that plays several crucial roles in the body. Its uptake is mediated by the taurine transporter (TauT). Genetic mutations and dysregulation of TauT have been linked to various neurological disorders, cardiomyopathy, childhood progressive retinal degeneration, and cancer, making TauT a promising target for therapeutic intervention in these diseases. However, the structure and mechanism of TauT remain poorly understood. In this study, we present the structures of the human taurine transporter (hTauT) under four conditions: the substrate-free state, the taurine-bound state, the β-alanine-bound state, and the cyclic inhibitor piperidine-4-sulfonate (P4S)-bound state. These structures reveal that taurine binds at the central substrate-binding site of hTauT. Notably, β-alanine and the cyclic P4S inhibitors also mimic taurine, occupying the same substrate-binding site. In the substrate-free and P4S-bound forms, hTauT also adopt an inward-open conformation, where transmembrane helix TM1a bends toward the membrane, facilitating the opening of the intracellular gate for ion and substrate release. These structural insights enhance our understanding of the mechanisms underlying substrate and ion recognition and transport in hTauT, paving the way for the future development of taurine transporter substrate analogues or selective inhibitors.

Introduction
Taurine (2-aminoethanesulfonic acid) is an abundant nonprotein β-amino acid and a conditionally essential nutrient widely distributed in most tissues of humans. Taurine has various physiological functions, including energy metabolism, osmoregulation, antioxidation, and neuromodulation 1. Taurine deficiency causes functional impairments in the central nervous system, cardiovascular system, immune response, skeletal muscle, and eye 1-5. The intracellular high levels of taurine are mainly regulated by the taurine transporters (TauT), which take taurine into cells 6. TauT-knockout mice exhibit aging characteristics, such as exercise intolerance and muscle atrophy, retinal and inner ear degeneration, renal development and function alteration, and cardiomyopathy 7,8. Mutations in the TauT genes are associated with neurological disorders, cardiomyopathy, and childhood progressive retinal degeneration in humans 9. In addition, TauT is overexpressed in several malignancies, such as colorectal (CRC), gastric (GC), and cervical cancers (CC), causing poor prognosis, immune evasion, aggressive and invasive progression, and recurrence of tumors 10-12. Therefore, TauT could be a promising diagnostic imaging and therapy target 13,14.

TauT (also known as SLC6A6) is a member of the solute carrier 6 (SLC6) family, which is divided into four subfamilies according to their substrate similarity—GABA (γ-aminobutyric acid), monoamine, neurotransmitter amino acid, and nutrient amino acid transporters. TauT shares >36% sequence similarity with other SLC6 family members, and has the highest sequence identity with GAT3 (~61%) 15. TauT, along with GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), BGT1 (SLC6A12), and CT1 (SLC6A8), belong to the GABA transporter subfamily 13. SLC6s can utilize the chemiosmotic gradient of sodium (Na+) and chloride (Cl-) ions to transport their substrates across the membrane 15. For TauT, substrate uptake occurs with a stoichiometry of 2 Na⁺:1 Cl⁻. Today, the substrate taurine of TauT has been approved for treating congestive heart failure 1. Taurine also shows potential in acting as a therapeutic agent to treat muscle diseases 16, neurological disorders 17,18, cardiovascular diseases 19, metabolic diseases 18, and cancer 20. Inhibition of TauT could be a promising strategy in cancer treatment, yet only limited inhibitors that are structurally similar to substrate have been investigated. β-alanine is a linear inhibitor of TauT, previously identified as the substrate of TauT to take up with high efficiency in neuronal and astroglial cells 21,22. Piperidine-4-sulfonate (P4S) is a cyclic inhibitor of TauT developed with the amine group incorporated into the ring 23. P4S shows a relatively higher affinity than other investigated inhibitors and provides a potential lead structure to design specific drug candidates of TauT for therapy of cancer and neurodegenerative diseases 23.

Understanding how substrate binds to and is transported by TauT and inhibitors act on TauT are critical questions in the field of TauT. The taurine binding site on TauT was mapped based on the structure of Aquifex aeolicus LeuT, and mutations of several residues have been shown to significantly influence substrate recognition and uptake function of TauT 24. Moreover, homology modelling of TauT based on the related subfamily member GAT1 structure has provided valuable insights into the protein-ligand interactions 13. However, the structure of TauT remains unknown, precluding a thorough understanding of its transport mechanism and the structure-based development of medications.

Here, we show cryo-EM structures of human TauT (hTauT) in the apo, taurine-bound, β-alanine-bound, and P4S-bound states in the inward-occluded conformation at resolutions ranging from 2.7 to 2.9 Å, respectively. The apo state and the P4S-bound state also show inward-open conformations. These structures reveal the overall architecture, the linear substrate- and ion-binding sites, the conformational transition of hTauT, as well as the inhibitory mechanism of the cyclic P4S.

Results
Structural determination and architecture of hTauT
Full-length wild-type human TauT (hTauT) was expressed with a green fluorescent protein (GFP) and maltose binding protein (MBP) at its N-terminus to monitor the behavior of hTauT protein via fluorescence-detection size-exclusion chromatography (FSEC) 25,26 (Supplementary Fig. 1a). hTauT with the GFP-MBP tag showed good behavior and thermostability when solubilized in the n-dodecyl-β-d-maltoside (DDM) + cholesteryl hemisuccinate (CHS) buffer (Supplementary Fig. 1a). A subsequent SEC-based thermal shift assay using purified proteins quantified this stabilization, showing the tagged hTauT had a melting temperature (Tm) of 82°C, significantly higher than the 46°C observed for untagged hTauT (Supplementary Fig. 1a). Therefore, full-length wild-type hTauT with the GFP-MBP tag was solubilized and purified in the DDM + CHS buffer with 150 mM NaCl throughout the solubilization and purification, owing to the essential roles of Na+ and Cl− ions in driving substrate uptake in SLC6s 15 (Supplementary Fig. 1b). The purified hTauT proteins were supplemented with different ligands for subsequent cryo-EM single-particle analyses (Supplementary Figs. 2-5). Initial single-particle processing of the apo and P4S-bound datasets indicated conformational heterogeneity, particularly in the TM1a helix, as evidenced by its discontinuous and poorly resolved density. This suggested significant flexibility, contrasting with the well-defined TM1a density observed in the stable, substrate-bound (taurine/β-alanine) states. To resolve this heterogeneity, we employed 3D variability analysis (3DVA) in cryoSPARC, which directly visualized the substantial flexibility of TM1a (Supplementary Movies 1-2). Subsequent focused 3D classification and refinement successfully separated two distinct conformations from these datasets: inward-occluded and inward-open (Supplementary Figs. 2 and 5). This strategy enabled us to determine high-resolution structures of hTauT in multiple conformational states: the apo state in the inward-occluded conformation and the inward-open conformation (2.8Å and 3.3 Å, respectively), the taurine-bound state in the inward-occluded conformation at 2.9 Å, the β-alanine-bound state in the inward-occluded conformation at 2.7 Å, and the P4S-bound state in the inward-occluded conformation and inward-open conformation (2.8 Å and 3.2 Å, respectively) (Supplementary Figs. 2-5 and Supplementary Table 1).

The qualities of these maps were all sufficient for model building of hTauT (Fig. 1 and Supplementary Fig. 6 and Supplementary Table 1). hTauT adopts the canonical LeuT fold with 12 transmembrane helices (TM1-12), resolved with clear side chains (Fig. 1, and Supplementary Fig. 6). Among the 12 TMs of hTauT, TM1-TM5 and TM6-TM10, two structurally similar but topologically inverted repeats (IRs), form the transporter core (Fig. 1a), which resembles the canonical structure of other transporters in the SLC6 family 27,28. TM1 and TM6 are two broken short helices (TM1a-1b and TM6a-6b) (Fig. 1). TM11 and TM12 settle around the transporter core, and TM12 has a pronounced kink in the middle of the membrane (Fig. 1). Noticeably, in the taurine- and β-alanine-bound states, TM1a is highly stable with clear N-terminal densities from P39. For the apo and P4S-bound states, conformational flexibility of TM1a—initially evidenced by discontinuous density—was resolved through 3D variability analysis and focused 3D classification as described above, yielding four well-defined structures. Among these, TM1a is ordered from P39 in the P4S-bound inward-occluded, S45 in the P4S-bound inward-open, and E42 in the apo inward-open conformations. An exception is the apo inward-occluded structure, in which the N-terminal density for TM1a begins at residue F50 (Supplementary Fig. 6 and Supplementary Table 1).

The extracellular surface of hTauT is formed by the extracellular loops (EL2, EL3, EL4, and EL6) (Fig. 1a-b). Distinctly, the large EL2 (residues 154-217) has clear densities except for a small region (residues 180-189). A conserved disulfide bond formed between C162 and C171 stabilizes the base of EL2 (Fig. 1 and Supplementary Fig. 6). Both N/C terminuses of hTauT extend into the cytoplasm. 602–620 of the C-terminus were not resolved, probably due to the high flexibility of these regions (Supplementary Fig. 7). The intracellular surface is composed of the N/C terminuses and intracellular loops (IL3, IL4, and IL5) (Fig. 1a-b). The C terminus of hTauT (residues 568-620) is formed with two visible helices (designated CH1–2) (Fig. 1 and Supplementary Figs. 6 and 7), that have various interactions with IL1, IL3, and IL5 (Fig. 1b). This is in line with the important regulatory roles of the C terminus of hTauT in its surface expression and uptake activities 6,29.

The cryo-EM maps revealed several lipid-like densities surrounding the hTauT transmembrane domain (Fig. 1a, c-e). We identified that three densities occupy positions analogous to the previously characterized CHOL2 and CHOL3 sites in related SLC6 transporters 30, as well as an additional identified CHOL4 site in hDAT (Supplementary Fig. 8) 31. These three densities show the characteristic shape and size of sterols, which were modeled as cholesterol or CHS molecules (Supplementary Fig. 8). Notably, the conserved CHOL1 site, which stabilizes the outward conformation in other SLC6 transporters 30, was unoccupied in all our hTauT structures (Supplementary Fig. 8), consistent with our structures representing inward conformations.

Taurine-binding mechanism of hTauT
To measure the transporting activity of hTauT, we performed the substrate uptake assay using the LC/MS-MS method, as reported previously 32. The tagged hTauT exhibited taurine transport with a Km of 30.5 µM, comparable to the 24.8 µM measured for untagged hTauT (Fig. 2a and Supplementary Fig. 9a), and consistent with the previously reported value (Km = 37.3 µM) measured using the GFP-tagged hTauT in HEK293 cells 23. This confirmed that the fusion tag does not impair the transporter's fundamental function. To investigate the structural basis of taurine recognition by hTauT, we determined the structure of the hTauT-taurine complex at 2.9 Å (Supplementary Fig. 3). A strong density was observed at the substrate binding pocket in the taurine-bound state (Fig. 2b, c), while no visible density was observed in the apo state of hTauT, as no substrate was added during the apo sample preparation (Fig. 2c). The shape and size of the density match that of the taurine molecule, suggesting that this density represents the taurine substrate (Fig. 2c). The taurine coordinates into the substrate binding site of hTauT, surrounded by TM1, TM3, TM6, and TM8 (Fig. 2d), as that of SLC6 family members 32-34. The bound taurine is completely occluded from solvent access (Fig. 2b). Furthermore, the first turn of the intracellular side of TM5 is unwound at the conserved G(X9)P motif (G243(X9)P253 in hTauT), hydrating the Na2 site (Fig. 2b). These features are hallmarks of the inward-occluded conformation, as seen in other SLC6 transporters like hGAT1 35 and MhsT 36 (Supplementary Fig. 9b), confirming that taurine-bound hTauT is trapped in the inward-occluded conformation.

The substrate binding site of MATs is divided into the trilobed subsites (A, B, and C) 30. The subsite A of MATs is delimited by TM1b, TM6, and TM8 30. The sulfonic acid moiety of taurine deeply penetrates an enlarged subsite A of hTauT (denoted as a subsite AL), in which the relatively large negative aspartic acid on TM1b of MATs (D75 in hNET) is replaced by a small glycine (G60 in hTauT) (Supplementary Fig. 10c). This altered subsite organization, which has previously been observed in related transporters hGAT1 35 and rGAT1 32, creates a distinct binding architecture (Supplementary Fig. 10). Various interactions are formed between the sulfonic acid group of taurine and the expanded subsite AL of hTauT. The sulfonic acid group of taurine denotes multiple hydrogen bonds with the main chain amino groups of G60 and G62 on the flexible hinge region of TM1. Meanwhile, the sulfonic acid group forms several polar interactions with side chains of Y138 on TM6 and S402 on TM8 (Fig. 2d). Moreover, one oxygen atom of the sulfonic group of taurine coordinates Na at the Na1 site, which is further stabilized by chelating with N63 on TM1 (Fig. 2d). The hydrophobic ethane group of taurine protrudes into the subsite B lined by the hydrophobic residues L134 and Y138 on TM3, F300, A303, and L306 on TM6 (Fig. 2d). The terminal amino moiety of taurine establishes a salt bridge with the side chain of E406 on TM8 in subsite C and also forms hydrogen bonds with the main chain carboxylate group of F300 on TM6 and a water molecule (Fig. 2d). This is consistent with previous findings showing that G57E completely lost the activity, and F58I, L306Q, and E406C mutations reduced both the taurine uptake and binding affinity 24.

We next sought to understand the determinants of hTauT's specificity by comparing it with another taurine transporter, the proton-coupled amino acid transporter 1 (PAT1, SLC36A1). Sequence alignment and structural comparison with an AlphaFold3-predicted hPAT1 model reveal both common and distinct features (Supplementary Fig. 9c, d). Key small residues that form the enlarged subsite AL in hTauT (G57, G60, G62, S301) are completely conserved in hPAT1 (G59, G62, G64, S268), suggesting a shared mechanism for accommodating the taurine sulfonate group (Supplementary Fig. 9c, d). Furthermore, F300 and E406 in hTauT, which interact with the amino group, are conservatively replaced with F267 and Q350 in hPAT1 (Supplementary Fig. 9c, d). However, other critical residues are not conserved (Supplementary Fig. 9c, d). Most notably, F58—a unique determinant in hTauT that coordinates the Na1 site—is divergent in hPAT1 (Supplementary Fig. 9c, d). This difference likely contributes to hPAT1's broader substrate tolerance and its characteristically low affinity for taurine (Km > 1 mM), contrasting with the high-affinity, selective transport by hTauT 37.

A comparative analysis across the SLC6 family reveals how the conserved LeuT fold has been evolutionarily adapted. The substrate binding modes were compared among taurine to hTauT, GABA to hGAT1 (PDB: 7Y7W, GABA transporter subfamily) 35, glycine to hGlyT1 (PDB: 8WFI, neurotransmitter amino acid transporter subfamily) 38, and noradrenaline to hNET (PDB: 8ZPB, MAT subfamily) 39 in their occluded conformations (Supplementary Fig. 10). Noticeably, the binding mode of taurine to hTauT closely resembles that of GABA to hGAT1 (Cα RMSD of 1.627 Å) and glycine to hGlyT1 (Cα RMSD of 2.535 Å) (Supplementary Fig. 10a, b). A key distinction, however, is that the taurine sulfonate group (with three oxygen atoms) engages in a more extensive hydrogen-bonding network than the carboxylate groups (with two oxygen atoms) of GABA or glycine. Moreover, several unique residues in hTauT (G57, F58, E406) are substituted in hGAT1 (Y60, A61, T400), explaining their distinct substrate profiles and the low affinity of hTauT for GABA 40. In contrast, hMATs like hNET have evolved a fundamentally different binding site configuration 41. The electronegative catechol moieties of monoamines are profoundly extended into the enlarged subsite B lined by the glycine on TM1b (E406 in hTauT), while their electropositive amino groups are anchored in a smaller subsite A by the large acidic residues (D75 in hNET), instead (Supplementary Fig. 10c). This configuration is inverse to that of hTauT, which features a small glycine (G60) in subsite AL and a large, acidic residue (E406) in subsite C (Supplementary Fig. 10c). This comparison underscores the remarkable versatility of the SLC6 scaffold, which has been tailored through specific residue changes to transport chemically diverse substrates.

The binding of substrate β-alanine to hTauT
TauT was originally identified as a taurine/β-alanine transporter, which can transport both taurine and β-alanine and similar Km values were measured using HEK293 cells (37.3 and 44.5 μM for taurine and β-alanine, respectively) 23. We confirmed that β-alanine acts as a competitive inhibitor of taurine uptake, with a Ki of 55.0 µM (Fig. 3a). To explore the binding mode of β-alanine, we resolved the structure of β-alanine-bound hTauT at 2.7 Å (Supplementary Fig. 4). Further inspection of the cryo-EM map revealed an isolated and prominent density at the substrate binding site, which aligns well with the structure of the β-alanine molecule (Fig. 3b, c). The binding pocket is blocked from both the extra- and intracellular sides and the intracellular side of TM5 is unwound, indicating that the β-alanine-hTauT complex also adopts an inward-occluded conformation (Fig. 3b). Similar to taurine, β-alanine also has a basic amine group and an acidic moiety connected by a linear two-carbon linker. The acidic moiety of β-alanine is a carboxy group instead of the sulfonic group in taurine. Two oxygen atoms of the carboxy group are also penetrated into the extended subsite AL and form multiple hydrogen bonds with residues with hTauT (Fig. 3d). One oxygen atom coordinates with Na+ at the Na1 site to bridge the interaction with F58 and N63 on TM1, and S301 on TM6. The other oxygen forms hydrogen bonds with the main chain amino group of G62 on TM1 and the side chain hydroxyl group of Y138 on TM3 (Fig. 3d). Additionally, the ethane group linker and the amino group of β-alanine form the same interaction as that of taurine with hTauT: the two-carbon linker is nested into the subsites C lined by the hydrophobic side chain of L134 and Y138 on TM2, F300, A303, and L306 on TM6, and the amino group of β-alanine also forms a hydrogen bond with the side chain of E406 on TM8 and water-mediated interactions with the main chain carboxylate group of F300 on TM6 and the side chain of Q403 on TM8 (Fig. 3d). Therefore, the interactions of β-alanine with hTauT are almost the same as those of taurine with hTauT; only the hydrogen bond network of the carboxy group of β-alanine with hTauT is less than that of taurine with hTauT, which has three oxygen atoms from the sulfonic group (Fig. 3e). This is in agreement with the functional analysis that hTauT has a higher affinity to taurine than β-alanine 42.

The binding of cyclic inhibitor P4S to hTauT
Different from β-alanine, P4S is a cyclic substrate analog of taurine containing one acidic and one amino head group linked by carbon chains 23. P4S acts as a competitive inhibitor of taurine transport by hTauT 23 and can dose-dependently inhibit the taurine uptake of hTauT with a Ki of 1.5 mM (Fig. 4a). To elucidate the binding mode of P4S with hTauT, we determined the P4S-bound hTauT complex structure in the inward-occluded conformation at 2.8 Å resolution (Fig. 4b and Supplementary Fig. 5). A distinct P4S density was observed at the substrate-binding pocket of hTauT (Fig. 4c). Similarly, the sulfonic acid group of P4S protrudes into subsite AL and forms multiple hydrogen bonds with the main-chain amino group of G60 and G62 on TM1a, the side-chain hydroxyl oxygen of Y138 on TM3, and S402 on TM8 (Fig. 4d). Furthermore, one oxygen atom of the sulfonic group of P4S coordinates Na+ at the Na1 site, identically to that of taurine (Fig. 4d). However, the rigid piperidine ring of P4S forms a boat conformation to fit less optimally into the hydrophobic subsite C of the substrate binding site. The piperidine ring confers relative rigidity to the nitrogen group, in which the interaction of nitrogen with E406 is disrupted (Fig. 4e). This is in agreement with the fact that the P4S is not a high-affinity inhibitor of hTauT with a high Ki. To validate the functional role of these residues at the P4S binding site, we designed a series of mutations (Fig. 4f). Surface expression assays revealed that most mutants were properly trafficked, with the exception of G62Y, which showed reduced expression (Supplementary Fig. 11a). As most residues in the P4S-binding site are also involved in substrate binding, mutagenesis of key binding site residues abolished uptake activity (Fig. 4f). Notably, the E406G mutation, which disrupts a critical salt bridge for taurine binding, resulted in a complete loss of function, underscoring its essential role (Fig. 4f).

Interestingly, besides the inward-occluded conformation, we also captured hTauT in an inward-open conformation in the presence of P4S at 3.2 Å resolution (Figs. 1, 5a, Supplementary Fig. 5 and Supplementary Table 1). The P4S-bound inward-open structure is highly similar to the apo inward-open structure with a root-mean-square deviation (RMSD) of 0.576 over 540 aligned Cα atoms (Supplementary Fig. 11b). In both the P4S-bound or apo inward-open structure, a pronounced bend of TM1a towards the membrane plane, coupled with unwinding of the intracellular side of TM5, collectively open a solvent-accessible pathway to the cytoplasm (Fig. 5b, Supplementary Fig. 11b). These structural features are consistent with the inward-open conformation of related transporters hGAT1, hGAT3, and hGlyT1, but distinct from the outward-tilted TM5 helix observed in the inward-open LeuT (Supplementary Fig. 11c) 35,43-45. In the substrate-binding site, there is a clear, well-defined density in the P4S-bound state, but does not exist in the inward-open conformation of the apo state (Fig. 5b). The density is sufficient to assign the P4S molecule. The P4S’s configuration is nearly identical between the inward-occluded and inward-open conformations (Fig. 5c). In the inward-open conformation, the sulfonic acid group of P4S remains engaged with subsite AL of hTauT and forms four hydrogen bonds with the main-chain amino group of G62 on TMl, the side-chain hydroxyl oxygen of Y138 on TM3, S301 on TM6 and S402 on TM8 (Fig. 5c). The principal conformational changes are the ~54° outward splaying of TM1a and further unwinding of the intracellular side of TM5 in the inward-open conformation (Fig. 5d). The movement of TM1a directly severs the hydrogen bond between P4S and G60 on TM1a. Furthermore, it disrupts the Na1 site, abolishing the Na⁺-mediated interactions with F58 and N63 on TM1a (Fig. 5c, d). Thus, while P4S remains bound in the inward-open conformation, its interactions with the transporter are partially destabilized due to the displacement of TM1a. This mechanism, wherein a substrate or substrate analog is retained in a partially weakened state during the gating transition, mirrors observations from noradrenaline-bound hNET structures 39. A comparison of substrate-binding modes in the inward-open conformation further highlights the divergent strategies employed by SLC6 subfamilies. Consistent with our analysis of the inward-occluded conformation, the binding architecture of hTauT is inverted relative to monoamine transporters. In the inward-open hNET, the electronegative catechol moiety of noradrenaline penetrates an enlarged subsite B, while its electropositive amino group is anchored in subsite A by a large acidic residue (D75). In stark contrast, hTauT employs a small glycine (G60) in subsite AL to accommodate the sulfonate group and a large acidic residue (E406) in subsite C to coordinate the amino group (Supplementary Fig. 11d). This conserved, inverted binding motif underscores a fundamental structural determinant of substrate specificity that is maintained throughout the transport cycle.

Conformational equilibrium of apo and P4S-bound hTauT
Following 3DVA and focused 3D classification, we resolved two distinct inward conformations of apo hTauT (Fig. 1a, b). The inward-occluded conformation, reconstructed from 149,606 particles, features an occluded substrate-binding site and a partially unwound intracellular segment of TM5 (Supplementary Fig. 12a). This conformation is nearly identical to the taurine-bound inward-occluded state, with a RMSD of 1.250 Å over 542 aligned Cα atoms (Supplementary Fig. 12b). In contrast, the inward-open conformation, determined from 80,335 particles (~35%), is virtually superimposable with the P4S-bound inward-open structure (RMSD 0.576 Å; Supplementary Fig. 11b). A comparison between the two apo conformations reveals that the transition to the inward-open conformation is characterized by a ~62° outward splaying of TM1a and further unwinding of the intracellular side of TM5, which opens a cytoplasmic pathway (Fig. 6a). The particle distribution (~65% inward-occluded vs. ~35% inward-open) in the apo state indicates a dynamic equilibrium with a preference for the inward-occluded conformation. The coexistence of both conformations suggests the apo transporter samples a dynamic conformational ensemble, which likely includes outward conformations. We therefore propose that apo hTauT exists in a dynamic equilibrium; when it transiently visits an outward-open conformation, taurine binding primes the transporter for translocation. This intrinsic conformational plasticity is a shared feature among SLC6 transporters, such as GlyT1 and SERT 44,46.

Strikingly, P4S binding reinforces this equilibrium. In the P4S-bound state, the majority of particles (~80%) populate the inward-occluded conformation, with a minority (~20%) in the inward-open state. This pronounced population shift demonstrates that P4S binding preferably stabilizes the inward-occluded conformation, which is structurally rationalized by the enhanced ligand-protein interactions observed in this state (Fig. 5d).

Structural change of hTauT from the occluded conformation to the inward conformation
To reveal how hTauT transits from the inward-occluded conformation to the inward-open conformation to release the taurine substrate into the cytosol, we next compare the taurine-bound hTauT structure (inward-occluded) with the apo hTauT structure (inward-open conformation). The structure of hTauT-apo can be superimposed to that of taurine-hTauT with a root-mean-square deviation (RMSD) value of 3.321 Å over 543 aligned Cα atoms (Fig. 6b). Most of the structures between these two conformations are the same except TM1a and further unwound TM5, which will be discussed later (Fig. 6b). Above the substrate binding site of both the inward-occluded and inward-open conformations, various interactions are formed among residues from the tightly packed TM1b, TM3, TM6a, and TM10 to close the extracellular pathway (Fig. 6c). F300 on TM6a interacts with Y138 on TM3, and R66 on TM1a forms a salt bridge with D459 on TM10 (Fig. 6c), which are two highly conserved EC-gate pairs in the SLC6 family for the closure of the EC gate 30. In addition, R66 forms a cation-π interaction with F300 and a hydrogen bonding network with the surrounding residues L61, L70, Q297, D459 and S464 to stabilize the closed EC-gate (Fig. 6c). R66 contributes two hydrogen bonds to D459 and one to Q297 and S464, respectively. The extensive interactions observed alongside the extracellular vestibule of hTauT are reminiscent of the inward-occluded structures of GAT135. A notable conformational change is TM1a between these two conformations (Fig. 6b). TM1a of the inward-open conformation unwinds the last helical turn (56-GGFVG-60) and bends approximately 53° towards the membrane (Fig. 6b), resulting in an open pathway from the central pocket to the cytoplasm. Below the substrate binding site of the inward-occluded conformation, the TM1a occludes the permeation pathway of the substrate to the cytosol. The main chain of G57 on TM1a forms a hydrogen bond with S402 on TM8, while G57 in TM1a of the inward-open conformation swing away from the central pocket, resulting in an open pathway from the central pocket to the cytoplasm (Fig. 6d). Various interactions lead to a tightly packed substrate pocket with a relatively stable TM1a in the inward-occluded conformation of the taurine/β-alanine-bound states. Three IC gate pairs are formed to stabilize the inward-occluded conformation further (Fig. 6d), which are conserved in the SLC6 family 30. W44 on TM1a forms a π-π stacking with Y315 on TM6. Two salt bridges are formed between R41 on TM1a and D416 on TM8 and K47 on TM1a and D325 on TM7, respectively (Fig. 6d). Meanwhile, R41 forms a hydrogen bond with Y315 on TM6.

hTauT plays an important role in osmoregulation to control cell volume; the Na+/Cl− gradient is utilized to determine taurine intake 6. Our high-quality cryo-EM maps enabled us to identify the binding sites of these ions. In the inward-occluded taurine-hTauT structure, Na1 is coordinated by the sulfonic oxygen of taurine, the carbonyl oxygen of F58 on TM1, S301 on TM6a, the side-chain carbonyl oxygen of N63 on TM1b, and the amide of N333 on TM7 (Fig. 6e), and the Na2 site is coordinated by the main-chain carbonyls of G56 and V59 on TM1 and L398 on TM8, as well as the side-chain oxygens of D401 and S402 on TM8 (Fig. 6f). The Cl− site is coordinated by the side-chain hydroxyls of Y83 on TM1, S301 on TM6a, and S337 on TM7, as well as the side-chain amides of Q297 on TM6a in both the inward-occluded conformation of taurine-bound state and inward-open conformation of P4S-bound state (Fig. 6g, Supplementary Fig. 11e). However, in the central substrate-binding pocket of the inward-open conformation of the apo state, the densities at the Na1, Na2 and Cl- sites were all absent (Fig. 6e, f, g). Compared with the inward-occluded state, the local structural organization of the Na1 and Na2 sites in the inward-open state has notably changed owing to the conformational change of TM1a. The carbonyl groups of G56, F58 and V59 on TM1a swing away from the Na1 and Na2 sites, respectively (Fig. 6e, f), resulting in the disrupted sodium-binding sites. Integrating these findings with previous studies demonstrates that the inward-occluded SLC6 transporters chelate the Na+ and Cl- ions for substrate binding; in contrast, inward-open SLC6s have disrupted Na1 and Na2 sites, facilitating the release of sodium and substrate into the cytosol 35,47. The absent density for Cl- in the inward-open apo state probably owing to its lower resolution or chloride release in this state.

Our structural data demonstrate that P4S can bind to both the inward-occluded and inward-open conformations of hTauT, analogous to how noradrenaline binds to multiple conformations of hNET33. This multi-state binding, its structural analogy to taurine, and its known role as a competitive inhibitor suggested it might itself be a transported substrate. To test this hypothesis, we performed a decisive efflux assay 48,49. P4S induced the efflux of preloaded [3H]taurine from HEK293-hTauT cells. Crucially, pretreatment with monensin (which elevates intracellular [Na+]) significantly augmented P4S-induced efflux (Fig. 7a). This potentiation is a key functional characteristic consistent with SLC6 substrate translocation, as it indicates a Na⁺-coupled exchange process, rather than the action of a simple, static inhibitor. In order to further elucidate whether P4S could be transported as a substrate or acts as an orthosteric inhibitor, we sought to compare the structure of the P4S-bound hTauT with the structures in other conformations with the full transport cycle. Because the outward conformation structure of hTauT was not available, we utilized the structures of the PF-03463275-bound hGlyT1 (outward-open conformation) 38 as a substitute. hTauT and hGlyT1 share high sequence similarity and similar occluded and inward-open conformations (Supplementary Fig. 7) and are likely to own a similar outward conformation. We aligned these structures based on the motifs near their substrate binding site (Supplementary Fig. 12c). From the inward-occluded conformation to the outward-open conformation, TM1b and TM6a move away from taurine/β-alanine and P4S (Supplementary Fig. 12c), while from the inward-occluded conformation to the inward-open conformation, TM1a moves away from both P4S and the physiological substrates taurine/β-alanine (Supplementary Fig. 12d). Therefore, the structures in the different conformations during the transport cycle are fully compatible with the binding of P4S, the same as that of taurine/β-alanine (Fig. 7b). Therefore, collectively, the functional efflux data and structural analysis provide conclusive evidence that P4S is not merely an orthosteric inhibitor but a translocatable substrate analog that competes with taurine for binding and undergoes the full Na⁺-coupled transport cycle.

Methods
Cell culture
HEK293F suspension cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 medium (Thermo Fisher Scientific) supplemented with 1% FBS at 37 °C with an atmosphere of 5% CO2 and 70% humidity. HEK293T adherent cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS at 37 °C with 5% CO2. Expi 293F suspension cells (Thermo Fisher Scientific) were cultured in 293F Hi-exp medium (Shanghai OPM Biosciences). Sf9 insect cells (Thermo Fisher Scientific) were grown in SIM SF medium (Sino Biological) at 27 °C.

Expression constructs and detergents screening
The complementary DNA (cDNA) of human TauT (1–620; UniProt: P31641) was purchased from HedgehogBio Science and Technology Ltd. (catalog number: HH-gene-1116). The cDNA of hTauT contains nine epitope transfer mutations (F58A, L306Q, E406G, G62Y, G60Y, L61A, L134A, S301A, G57A) were cloned into a modified BacMam expression vector 51 with the N-terminal GFP-MBP tag. GFP is fused with two strep tags in its loop for affinity purification. All primer sequences are provided in the Source Data file. The BacMam plasmids containing the gene of hTauT were small-scale transient transfected into HEK293F cells. The cells were collected after 48 h transfection and solubilized in different detergents to screen detergents and stability of hTauT via fluorescence-detection size-exclusion chromatography (FSEC) on the Superose 6 increase 5/150 GL column (Cytiva). Detergents were used at the following concentrations: 1% (w/v) DDM plus 0.1% (w/v) CHS, 1% (w/v) DDM, 1% (w/v) lauryl maltose neopentyl glycol (MNG), 1% (w/v) MNG plus 0.1% (w/v) CHS, 1% digitonin, and 1% (w/v) glyco-diosgenin (GDN).

Protein expression and purification
The baculoviruses of hTauT were produced with the Bac-to-Bac system and amplified in Sf9 insect cells. HEK293F cells were typically infected with a 7% (v/v) volume of infection of the P2 virus at a density of 2.5-3.0 × 106 ml−1. 10 mM sodium butyrate was added to the culture 8-12 h post-infection. Cells were collected 48 h post-infection by centrifugation at 3,500 g for 10 min and washed once with 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. The harvested cells were then flash-frozen and stored at −80 °C. The protein was extracted from cells by resuspending the pellet with 20 mM HEPES pH 8.0, 150 mM NaCl, 2 µg ml−1 aprotinin, 2 µg ml−1 pepstatin, 2 µg ml−1 leupeptin, 1 mM phenylmethanesulfonyl fluoride (PMSF), and solubilized with 1% (w/v) n-dodecyl-β-D-maltoside (DDM; Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS; Anatrace) at 4 °C for 1 h. The insolubilized cell debris was removed by centrifugation at 3,500 g for 15 min. The supernatant was obtained by ultracentrifugation at 100,000 g for 1 h and then loaded onto 5 ml of Streptactin Beads 4FF (Smart-Lifesciences). Wash buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 0.025% DDM, and 0.0025% CHS was used to remove contaminating proteins. The target protein was eluted with 20 mM HEPES pH 8.0, 150 mM NaCl, 0.025% DDM, 0.0025% CHS, and 2.5 mM desthiobiotin (IBA Lifesciences). After adding 1 mM DTT, 10 mM MgCl2 and 2 mM ATP to remove contamination of heat shock proteins, the eluted target protein was concentrated with a 50 kDa cut-off concentrator (Millipore) and further purified by the Superose 6 increase 10/300 GL column (Cytiva) running in a buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 0.025% DDM, and 0.0025% CHS. The peak fractions related to the target protein on SDS-PAGE and showing good properties on native-PAGE were combined and concentrated for cryo-EM sample preparation.

Thermal stability assay
Thermal stability assays were performed according to a previously established protocol 25 with modification. Tagged and untagged hTauT proteins (1 µg and 2 µg, respectively) were diluted in assay buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.025% (w/v) DDM, 0.0025% (w/v) CHS) to a final volume of 100 µL. The solutions were heated for 15 minutes at specified temperatures in a thermal cycler. Denatured protein was subsequently removed by ultracentrifugation at 98,400 × g for 20 minutes using a Type 42.2Ti rotor (Beckman Coulter). The resulting supernatants were analyzed by FSEC. The FSEC peak heights for hTauT were normalized to the value from an unheated control sample. Melting temperatures (Tm) were determined by fitting the normalized data to a sigmoidal dose-response model in GraphPad Prism 8.

Uptake assay
HEK293T cells were first seeded into a 6-well plate at a density of 2.0 × 106 cells per well and infected with 0.5% (v/v) P2 virus after culturing for 12 h. 24 h-post infection, HEK293T cells were trypsinized for 1 min and seeded into a poly-d-lysine-treated 96-well plate at 2.0 × 106 cells per well in 100 μL culture medium. The supernatant of the 96-well plate was discarded 36 h after infection, and the cells were washed three times with uptake buffer consisting of 119 mM NaCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 4.7 mM KCl, 11 mM glucose, and 25 mM HEPES in distilled water, adjusted to pH 7.4 using NaOH. Saturation experiments were conducted with seven concentrations of taurine-d4 ranging from 0.4 μM to 640 μM. For competition experiments, β-alanine ranging from 1 μM to 10 mM and P4S ranging from 10 μM to 100 μM were pre-incubated at 37 °C for 4 min and 15 min, respectively. Then, 5 µM taurine-d4 was co-incubated with the same concentration ranges of β-alanine and P4S for 1 min at 37 °C. The stated concentrations always refer to the final assay volume of 100 μL. Uptake was terminated by washing the plates three times with cold PBS buffer. Cells were lysed at 4°C for 2 h by adding 100 μL of lysis buffer per well, which consisted of 90% methanol and 10% distilled water (v/v), containing 1 μM taurine-13C2 as an internal standard. Plates were subjected to LC-MS/MS quantification of taurine-d4. Nonspecific uptake was determined analogously with HEK293T cells infected with 0.05% (v/v) P2 virus of empty vector. Transport kinetics were analyzed using GraphPad Prism 8 software by fitting substrate concentration–uptake rate response curves. In the saturation experiment, specific uptake was calculated as the difference between total and nonspecific uptake. Saturation curves were generated using nonlinear regression analysis with the equation y = Vmax × x / (Km + x). Values of both IC50 and Ki were obtained by data interpolation of % Inhibition and the equation IC50 = Ki (1 + [S] / Km), respectively.

The chromatographic separation was performed on a Thermo Vanquish liquid chromatograph (Thermo Fisher Scientific) with an OSAKA SODA CAPCELL PAK ADME column (2.1 mm × 100 mm, 3 μm) maintained at 35 °C. The mobile phases were water (A) and acetonitrile (B), both containing 0.1% formic acid at a flow rate of 0.2 mL min-1. The elution procedure was set as follows: 0-1.5 min, 2% B; 1.6-3.8 min, 85% B; 3.9-8.0 min, 2% B. The autosampler was kept at 15 °C. Mass spectrometry was carried out on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). The capillary temperature was set to 320 °C, and the spray voltage was +3,500 V. Positive ion analysis was carried out in the selected-ion-monitoring mode. The resolution, maximum IT, and AGC target values were set at 70,000, 100 ms, and 3e6. The monitoring ions were 130.04705 and 128.02866 for taurine-d4 and taurine-13C2, respectively. The raw data was acquired using Xcalibur 4.1.31.9 software (Thermo Fisher Scientific).

Cell surface biotinylation and immunoblot analysis
Cell surface biotinylation was conducted as described before 52 with modification. HEK293T cells were seeded in six-well plates at a density of 2.0 × 10⁶ cells per well and subsequently infected with baculovirus for the expression of untagged hTauT, GFP-MBP-tagged hTauT, or its mutants. The titer of the P2 virus for each sample was standardized based on total protein expression levels measured by FSEC. At 36 hours post-infection, cells were washed three times with ice-cold PBS (pH 8.0). Subsequently, 500 µL of 1.5 mM Sulfo-NHS-LC-biotin (APExBIO) in PBS (pH 8.0) was added to each well, and the plates were incubated at room temperature for 1 hour. The reaction was quenched by replacing the biotin solution with ice-cold 10 mM glycine in TBS (pH 8.0). After washing with TBS (pH 8.0), cells were lysed with 1% DDM in TBS (pH 8.0) for 40 min on ice, followed by centrifugation at 16,125 × g for 20 minutes. The supernatants were incubated with HRV-3C protease for 15 minutes at 4°C to cleave the GFP-MBP tag. The lysates were then incubated with Streptactin Beads 4FF (Smart-Lifesciences) for 2 hours at 4°C. The beads were collected by centrifugation at 1,000 × g, and the flow-through was discarded. After four washes with lysis buffer, bound proteins were eluted with elution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 3 mM biotin, 100 mM DDT, 2% SDS) for 7 minutes at room temperature. The eluates were treated with PNGase F in Glycoprotein Denaturing Buffer (400 mM DTT, 5% SDS, 10% NP-40 in TBS, pH 8.0) at 37°C for 1 hour and then separated on a 12% gradient SDS-PAGE gel. For immunoblot analysis, proteins were transferred to a membrane and probed with an anti-Na+/K+ ATPase antibody (1:20,000; Abcam; ab76020) followed by goat anti-rabbit secondary antibody (1:5,000; Lablead; S0101) as a loading control to normalize sample volumes. hTauT was detected using an anti-hTauT antibody (1:1,000; Santa Cruz; sc-393036) followed by goat anti-mouse secondary antibody (1:5,000; Lablead; S0100).

Radiolabelled taurine release assay
Release assay was performed as previously described 48 with modification. Expi293F cells were infected with 0.5% (v/v) P2 virus of hTauT and incubated for 24 h to reach a density of 3.0 × 10⁶ cells. The culture medium was removed, and cells were loaded with 10 nM [³H]taurine in Krebs-Ringer-HEPES buffer (KHB: 120 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 20 mM glucose, 10 mM HEPES, pH 7.3) for 20 minutes at 37°C. The cells were then equilibrated to room temperature and washed five times with KHB. Following a 10-minute pre-incubation in either KHB alone or KHB containing 10 µM monensin, the supernatant was discarded. The release protocol was initiated with four sequential 2-minute incubations in 100 µL of the corresponding buffer (KHB or KHB + monensin), with supernatant collection at each interval. Test compounds, dissolved in the same buffers, were then applied for five additional 2-minute cycles. Taurine (400 µM) was used as a positive control. Each condition was tested in duplicate across three independent experiments. Collected supernatants were mixed with a liquid scintillation cocktail, and radioactivity was quantified by scintillation counting.

Cryo-EM sample preparation and data acquisition
The purified wild-type hTauT protein was concentrated to about A280=20 (11 mg ml⁻¹) to prepare the apo samples and incubated at 4°C for 2 h with 2 mM taurine, 5 mM β-alanine, or 2 mM P4S separately to prepare substrate-bound samples and inhibitor-bound samples. Then, 2.5 µL of the sample was added to holey carbon grids (GIG-Cu 300 mesh, R 1/1) glow-discharged for 2 min using 25% O2 and 75% Ar by the Solarus advanced plasma system (Gatan). The sampled grids were blotted for 4 s with a blot force of 2 after a 5 s incubation on the grids at 4 °C under 100% humidity and then plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific).

A Talos Arctica electron microscope (Thermo Fisher Scientific) operating at 200 kV and equipped with a K2 camera (Gatan) was first used to screen the quality of samples on cryo-EM grids. Cryo-EM data were acquired on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K3 Summit direct electron detector (Gatan) mounted after a quantum energy filter set to a slit width of 20 eV.

For taurine-bound hTauT, movie stacks were collected using EPU software (Thermo Fisher Scientific) in super-resolution mode at a magnification of ×64,000 with a pixel size of 0.54 Å and a defocus range of -1.5 to -1.8 µm. Each movie stack consisting of 32 frames was exposed for 2.68 s, yielding a total dose of about 50 e- Å-2 with a dose rate of 21.8 e- pixel-1 s-1. For the apo state, β-alanine- and P4S-bound hTauT, movie stacks were obtained using EPU software (Thermo Fisher Scientific) in super-resolution mode at a magnification of ×81,000 with a calibrated pixel size of 0.5335 Å and a defocus range of -1.5 µm to -1.8 µm. Each movie stack consisting of 32 frames was exposed for 2.56 s, yielding a total dose of about 50 e- Å-2 with a dose rate of 22.36 e- pixel-1 s-1.

Cryo-EM data processing
The data-processing workflows are displayed in Supplementary Figs. 2-5 and Supplementary Table 1. A total of 1,216 multi-frame movies of taurine-bound hTauT were collected. The beam-induced motion was corrected and twofold binned to a pixel size of 1.08 Å with dose weighting applied using MotionCor2 53. After manually removing empty carbon and micrographs with ice or ethane contamination, the dose-weighted micrographs were then imported into cryoSPARC 54, and contrast transfer function (CTF) parameters were estimated using patch-CTF estimation 55. Blob picker was used for initial particle selection from 1,210 out of a total of 1,216 micrographs, followed by several rounds of 2D classification, resulting in the selection of 269,729 particles to generate a good initial model with obvious secondary structure features by ab initio reconstruction. With the help of the good model, particles from a total of 1,210 micrographs were re-picked using Topaz 56, and seed-facilitated 3D classification 57 was performed to improve resolution. After seed-facilitated 3D classification and multi-reference 3D classification, the final map reached a resolution of 2.9 Å using non-uniform refinement 58 followed by local refinement with the soft mask covering hTauT.

For the apo state, β-alanine- and P4S-bound hTauT datasets, 2,628, 1,008, and 6,381 super-resolution movie stacks were collected, corrected, and twofold binned to a pixel size of 1.067 Å with dose weighting applied using MotionCor2, and micrographs showing contamination and empty carbon were manually excluded. The dose-weighted micrographs were then imported into cryoSPARC, and CTF parameters were estimated using patch-CTF estimation.

After being picked by the blob picker, 1,820,272 particles of β-alanine-bound hTauT were selected for two rounds of 2D classification to generate a good initial model with obvious secondary structure features by ab initio reconstruction. Seed-facilitated 3D classification and multi-reference 3D classification were performed to further improve the resolution to 3.7 Å. Particles were re-picked using two rounds of Topaz, processed for seed-facilitated 3D classification and multi-reference 3D classification to improve the map quality. Non-uniform refinement followed by local refinement with the soft mask covering hTauT was used to obtain final maps with resolutions of 2.7 Å for the β-alanine-bound hTauT.

Particles of the apo and P4S-bound states were picked by the blob picker underwent several seed-facilitated 3D classifications and multi-reference 3D classifications using the good reference to obtain a good initial model with a resolution of 3.6 Å for the apo state and 3.7 Å for the P4S-bound hTauT generated by its own particles. Particles were re-picked using Topaz, processed for seed-facilitated 3D classification and multi-reference 3D classification to improve the map quality. Further processing of the apo and P4S-bound datasets still showed discontinuous and poorly resolved density of TM1a, which indicates conformational heterogeneity. To resolve this heterogeneity, we employed 3D variability analysis (3DVA) in cryoSPARC. A consensus refinement map, low-pass filtered to 6 Å, served as the initial reference. A soft mask was applied to focus the analysis on the dynamic TM1a region. The 3DVA was computed for 3 modes over a maximum of 20 iterations. Following this, particles were visualized and sorted using the 3DVA display tool with a windowing setting of 2, which organized them into 20 overlapping classes based on their position along the computed mode. This analysis directly visualized the substantial flexibility of TM1a (Supplementary Movies 1-2). Subsequent 3DVA and focused 3D classification successfully resolved this heterogeneity, distinguishing two distinct conformational states (the inward-open and inward-occluded conformations) for both the apo and P4S-bound hTauT. To further improve map quality, a second round of particle picking was performed with Topaz. The resulting particles were subjected to seed-facilitated and multi-reference 3D classification. For the inward-occluded conformation of both the apo and P4S-bound hTauT, non-uniform refinement followed by local refinement with the soft mask covering hTauT was used to obtain final maps with resolutions of 2.8 Å. To specifically improve the density for the dynamic TM1a region in the inward-open states, we applied focused 3D classification centered on this helix, followed by non-uniform and local refinement. This strategy yielded final maps at 3.3 Å for the inward-open conformations of the apo state and 3.2 Å for the P4S-bound hTauT.

Model building and refinement
The initial structure model of hTauT was computationally generated using AlphaFold3 (AlphaFold Protein Structure Database, #AF-P31641-F1) 59 and then fitted into the cryo-EM map using UCSF Chimera X 60 and rebuilt manually using Coot 61. Phenix.real_space_refine and phenix.validation_cryoem tools in PHENIX 62 were used to further refine and validate models to obtain final structures. The model-related metrics, including MolProbity scores, clash scores, rotamer outliers, and statistics of the Ramachandran plots, were summarized in Supplementary Table 1. Figures were displayed with PyMOL63 and ChimeraX. The sigma levels for all presented density figures were calculated in ChimeraX through division of the threshold level by the standard deviation (SD).

Quantification and statistical analysis
Global resolution estimations of cryo-EM density maps are based on the 0.143 Fourier shell correlation criterion. The local resolution was estimated using cryoSPARC. The figure legends detail the number of technical replicates (n), along with relevant statistical parameters for each experiment. No statistical methods were used to predetermine sample sizes. The cell lines were routinely checked to be negative for mycoplasma contamination but have not been authenticated.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Qi, Zhang, and colleagues present human taurine transporter (TauT) structures in different states, revealing how taurine, β-alanine, and a putative inhibitor cyclic P4S bind to the substrate-binding site. The authors’ findings provide key insights into TauT’s transport mechanism, aiding future drug development. The manuscript is well and clearly written and provides a wealth of data - mostly derived from cryogenic electron microscopy (cryo-EM) approaches. While the structural work is at a very high standard – it does not come along with biochemical controls as it would be required to support the findings. Just a few things that the authors may take into consideration exploring: 1) The authors use an N-terminal GFP-MBP tag to use this construct expressed in HEK293 cells in suspension; however, it has not been described whether the construct is fully functional. The uptake assay performed in Figure 2, panel A, does not give the details of whether this particular construct has been used (and there is a typo: wide-type instead of wild type hTauT). Maybe the authors have performed these experiments and published the findings in an earlier paper – then, they ought to simply cite the evidence. If not, they must present the functionality of TauT in this specific vector by an uptake assay since the assay is anyway already up and running. 2) The number of structures in occluded or inward-open conformation is impressive and the authors have made a very good job in deciphering all these. However, since structural biological answers aided by cryo-EM largely relies on assumptions and interpretations of data, it would be helpful if the authors would add data of mutagenesis experiments. The manuscript includes quite a number of very plausible amino acid contact points with substrates or inhibitors; hence, it would be an easy task to mutate some key residues to ascertain loss of function and thereby support the findings of the cryo-EM approach (I am especially focusing on Figure 4 for this request). 3) The question whether cyclic P4S is a substrate or inhibitor cannot be answered by the assay the group has used in this manuscript. This ought to be done in a trans-acceleration experiment or also by a release assay: here. tritiated taurine is being transported into the TauT-expressing cells, and then, unlabeled taurine or unlabeled P4S is being used in increasing concentrations. In case of taurine, the tritiated taurine will be released by the transporter upon the inward-transport by the taurine applied from the outside. However, the problem is that the efflux can be elevated but still not be decisively showing that the compound is a substrate because in some instances, pseudo-efflux may take place. Since TauT is a member of the SLC6-family and related to GABA-transporter, it is dependent on sodium. To determine the substrate status of P4S, the authors should use monensin, a sodium-proton ionophor, which will selectively elevate sodium ions intracellularly while depleting protons. This activity will then be decisive to unravel the substrate status of the compound: Only substrates will be enhanced in their efflux behavior while inhibitors will not show enhancement of efflux. This has been shown not only for members of the SLC6-MAT family but also for the GABA transporter GAT1. The authors shall follow these examples to clearly interrogate the substrate status of P4S. With these additional experiments, the already interesting and broad study will be lend support – and be even more powerful.", "reviewer_2": "The manuscript authored by Jing-Xiang Wu and colleagues reports the structures of TauT at a good resolution through cryoEM in complex with taurine, P4S, beta-ala and substrate free states in different conformations. While the study is technically sound, I have the following major concerns that affect the paper’s quality and raises questions that need to be answered. The overall structures of TauT appear to be of a good quality however the authors stop at just reporting the TauT interactions and conformational transitions that are generally in accordance with the other SLC6 members. There seems to be no attempt to study the molecule in depth by making mutations in and around the binding pocket and observing transport kinetics. There seems to be no particularly insightful observations for the mechanism and if there are any characteristics different from other SLC6 members. The conclusions are generally weak and really give no added insight into this transporter group. Further the density of P4S seems to be weak in the occluded state of the transporter. The authors need to give a contoured density separately of P4S. Have any binding sites of P4S observed in the inward-open state of the transporter since the structure in inward open state says that it is P4S inward in fig 1e. A more detailed, direct biochemical evidence is required to ascertain if P4S is indeed a transporter. It cannot be based on structural comparisons. The Authors do not seem to report the presence of any bound detergents or lipids despite achieving a high resolution. In most cases some cholesterol molecules tend to be visible when DDM CHS is used for structure determination. beta-alanine substrate competition and uptake could have been studied to provide more insights into the ability to compete with taurine. This was only done as a qualitative assay whereas a Ki measurement could have been done with beta-alanine. The subsite organization demonstrated here does not highlight altered organization observed in GATs in comparison to the MATs reported earlier. The proper literature for this subsite organization has to be cited as well. Overall the study study reports a set of TauT structures but the conclusions that could have been made through a detailed analyses of substrate recognition and transport mechanisms are lacking. Some more minor comments are given below. - Was the thermal stability assay done using the protein containing GFP-MBP? The higher melting point can be due to the presence of GFP and MBP tags. - Expression construct has no mention of strep tag but in purification, streptactin beads were used for affinity chromatography. - In native PAGE , there is a band which corresponds to MW between 130 to 180 kDa. Could it be some oligomeric species?", "reviewer_3": "The manuscript “Structural mechanism of substrate binding and inhibition of human taurine transporter” describes 6 cryo-EM structures of human taurine transporter (TauT, SLC6A6) bound to taurine, b-alanine, competitive inhibitor P4S, and in substrate-free states. This work is the first presentation of the therapeutically important target, the human TauT structures. While the cryo-EM density maps are of good quality, and the study provides interesting details of the transport cycle, the manuscript can benefit from more in-depth discussion of the findings, placing the study in the context of the wealth of structural and functional data available on SLC6 transporters. Notable issues: - There is no mention of TM5 unwinding and solvation in inward-open states, a characteristic of inward facing states of SLC6 transporters. Whether or not this can be observed, should be discussed. See the reference: Malinauskaite L, et al., doi: 10.1038/nsmb.2894. Epub 2014 Oct 5. PMID: 25282149 - The inward facing states presented in this study, are “inward-open” states, please correct the text accordingly. - Please elaborate on the occluded states, are they outward/inward occluded, or how do they compare to previous SLC6 occluded states, the most similar ones? 249 – 259 discusses this part to some extent but requires further elaboration. - Line 265: ‘and are alternative substrates’, and line 259: ‘This suggests that P4S might be an alternative substrate to hTauT’. This is too definitive based on the evidence presented in the manuscript as to whether P4S is an alternative substrate or substrate analogue. It has only been speculated from structural comparisons, but no functional experiments were carried out. Therefore, it would be best to be more speculative with this statement. - Binding site comparison of taurine-bound TauT with PAT1 (SLC36A1), another taurine transporter, on sequence level (and perhaps on a model basis – alphafold model) could be relevant. - The order of figures does not follow the order they are mentioned in the text, please correct that. - Uptake assay: It is not clear how many replicates were conducted in this assay and what data is being shown (mean? Single data point?). - Is there any indication that P4S gets transported by TauT, whether based on previous studies or experiments performed by the authors? - Local density figure, currently extended data figure 6, should be provided for all the models and maps presented in the manuscript, separately, and not just representative sections of selected models and maps. - Distances should be mentioned for the presented interactions on figures. - Label the TMs on figures, please. - The sigma levels are correctly reported for all the presented density figures; however, it is not clear if they are in reference to Chimera, coot, or? please specify that in the figure legends. - Line 250: The outward-open structure of GlyT1 is not substrate-free! It is bound to competitive inhibitor PF03463275. Models and maps: - There is no density for NAG sites, e.g., in the map 2638, please remove the NAGs from this model and others if the density is not supporting the modelling. - P4S-bound model – occluded state: The fit of the inhibitor could be optimised, the good quality of the density should allow a slightly better fitting. There appears to an interaction of nitrogen group of P4S with CO of E409, if the pose of P4S is modified to fit the density better. If that is the case, the pose should be optimised to see if the interaction is possible. - P4S-bound model – presented as the inward-open It is quite important to clarify whether the P4S also stabilises an inward-open state, or the density in binding pocket accommodates another small molecule. The general fit of the model into density is not finalised, the map is noisy. The noise could originate from the local refinement, if a mask has been used, and needs to be optimised/improved to be evaluated. - Substrate-free states: The substrate-free states can benefit from a section in the manuscript, clearly describing the two states, discussion on differences, if and how they were obtained. They are obtained from one data set, and it’s quite amazing considering the data set is not very large. Please elaborate on the two states, the how, the why, and descriptions in a separate section, similar to other states. In map 2630, there is density around the TM5 region that is not modelled. Could the authors comment on the density whether it is noise (that should be processed better) or if the TM5 can be partially modelled into that density? The map is still noisy, and the model fit at the turns/loop regions can clearly be improved. The disulfide bridge C162-C171 in EL2 seems to be broken in map 2638, please comment on this and other maps. - b-alanine-bound state: There is extra density in the binding pocket. It could nicely accommodate a water molecule and a water mediated interaction of b-alanine with E403. Please consider modelling the Na1 in the b-alanine-bound state, the density is not as strong, but resealable. - Taurine-bound state Extra density in the binding pocket of Taurine-bound state– could be again coordinating water molecule with the Nitrogen of the substrate. Ions binding site – Please add the density to ion binding site figures. Figure 1 – Please present all the models and respective maps at least once in the manuscript. Change ‘functional states’ to ‘conformational states’ a) Report the density level of the map used for the figure. e) hTauT is bound to P4S in two different conformations however P4S is not shown in the inward conformation as it was not fitted. It may be beneficial showing the density that you do observe in this part of the figure (please see above comments as this map/model needs further clarification). Figure 3a β-Alanine binding mode “by” hTauT. Replace “by” with at, or in. Next line as well: The inhibition of β-alanine “to” hTauT – also not clear what it means? Isn’t β-Alanine a substrate? It is not clear what this graph is showing and why it is presented as a bar plot. How many independent experiments were performed? If the authors are showing replacement of hot-taurine with β-Alanine, the data should be presented more clearly. Figure 5. Please outline how the degrees of motion were measured. Label all the TMs in the individual panels, not just TM1. Extended data figure 1. Outline the detergent concentrations used for the experiments conducted in panel a. The screening of detergents was not outlined in the Methods. Extended Data Table 1: Initial model used: please provide the respective alphafold model ID(s). Please add the CaBLAM values for the models (it should be below 1% to be acceptable). Extended data figure 9, g, the label of the top model is placed behind the figure. The figure can be expanded accommodating the various states identified in this study and perhaps should be a main figure. - Change SLC6A to SLC6 though the manuscript Line 30: development of “substrate”? substrate analogue perhaps? Or selective inhibitors. Line 35: Remove ‘The’ such that it reads ‘neuromodulation’. Taurine deficiency causes functional…’ Line 38: A reference to knock out mice studies should be added Line 48: Change shares to share Lines 49 – 51 don’t read well, e.g., belong to GABA transporter subfamily? Line 49: How much similarity is shared? Which member has the highest sequence identity? Line 51: A comment on the stoichiometry and mentioning of chloride would be beneficial. Line 52: Many members have overlapping substrates, and not all have unique substrates. Line 56: Change ‘substrate-structural-similar inhibitors’ to ‘inhibitors that are structurally similar to substrate’ Line 62: Change ‘substrate binds and transports by TauT’ to ‘substrate binds to and is transported by TauT’ Line 88-91: Be specific about the inward conformation. Not clear from the text if it is inward-open or inward occluded Lines 103 – 104 don’t read well. Lines 105 – 116: It is not clear what structure is being discussed here, if it is the general architecture, please clarify that. Line 142: It is not clear which residues are TM3 and which are TM6 in the use of ‘respectively’. Line 153: Remove ‘smallest’ Lines 188 – 190: It is not clear what is the purpose of this sentence, wasn’t this expected from the chemical structure of P4S? Line 191: Rephrase to outline which are main chain interactions and which are side chain interactions Line 255: Change ‘away taurine/…’ to ‘away from taurine/…’ Line 256: Rephrase. ‘TM1a moves away from P4S’. It is not clear what the authors are trying to communicate with ‘as taurine/β-alanine’ Line 282: BacMam is a virus expression system. ‘transfected’ should be replaced with ‘transduced’ or further explanation should be used if transfection was used for FSEC screening. Line 290: replace ‘solubilizing’ with ‘solubilized’ Line 292: replace rpm value with g value Line 345: replace ‘cryo-grids’ with ‘cryo-EM grids’ On the methods section: 278 – 282: It is a very long sentence starting with DNA and ending with protein expression, please break it into shorter sentences for clarity. Similarly, the next section: FreeStyle 293F cells at a density of 2.5-3.0 × 106 ml−1 were infected for 48 h with P2 virus produced with the Bac-to-Bac system using Sf9 insect cells, and 10 mM sodium butyrate was added to the culture 8-12 h post-infection. Can be modified as: The HEK293F cells were typically infected with a xx% volume of infection of the P2 virus (please add the titre) at a density of 2.5-3.0 × 106 ml−1. 10 mM sodium butyrate was added to the culture 8-12 h post-infection, and the cells were collected 48 h post-infection by centrifugation at …… Production of virus with Back-to-Bac system can be added as a line in the beginning. Please provide as much details as possible: Was the purification performed right after harvest? Or were the cells stored in -80 C? Line 299: Superdex 6 increase 10/300 – superose 6? Line 206: How much P2 virus? mention the titre please. Line 206: After infecting for 24 h, can be modified to: 24 h-post infection Line 351: “a defocus of -1.6 μm”, has it been only one defocus value for the taurine-bound hTauT data collection, and not a range? If so, please elaborate. The values also differ from the cryo-EM table, please correct accordingly. Line 342: flash-frozen --> plunge-frozen Line 387: please reference the non-uniform refinement article as recommended by cryoSPARC programmers Line 393: please provide the respective alphafold model ID(s).", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-to-point response to reviewer comments\nWe sincerely appreciate the editors and reviewers for their time and insightful\ncomments. Your valuable and constructive comments are of great importance in\nimproving our manuscript. As suggested, we have carefully revised our manuscript\nand performed additional experiments. We have now completed the revision and\naddressed all comments raised by reviewers. Please find the point-to-point responses\nbelow.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Qi, Zhang, and colleagues present human taurine transporter (TauT)\nstructures in different states, revealing how taurine, β-alanine, and a putative inhibitor\ncyclic P4S bind to the substrate-binding site. The authors’ findings provide key insights\ninto TauT’s transport mechanism, aiding future drug development. The manuscript is\nwell and clearly written and provides a wealth of data - mostly derived from cryogenic\nelectron microscopy (cryo-EM) approaches.\nResponse: We are very grateful to the reviewer for your positive and insightful\ncomments.\nWhile the structural work is at a very high standard – it does not come along with\nbiochemical controls as it would be required to support the findings. Just a few things\nthat the authors may take into consideration exploring:\n1) The authors use an N-terminal GFP-MBP tag to use this construct expressed in\nHEK293 cells in suspension; however, it has not been described whether the construct\nis fully functional. The uptake assay performed in Figure 2, panel A, does not give the\ndetails of whether this particular construct has been used (and there is a typo: wide-\ntype instead of wild type hTauT). Maybe the authors have performed these\nexperiments and published the findings in an earlier paper – then, they ought to\nsimply cite the evidence. If not, they must present the functionality of TauT in this\nspecific vector by an uptake assay since the assay is anyway already up and running.\nResponse: We thank the reviewer for raising this important point regarding the\nfunctionality of our tagged construct. As you suggested, we have compared the\ncomparative functional characterization between hTauT with the GFP-MBP tag and the\nuntagged hTauT.\nAs shown in the new Fig. 2a, we determined the transport kinetics using an LC/MS-\nMS-based uptake assay. The tagged hTauT exhibited a Michaelis constant (K ) of 24.8\nm\nµM, which is highly comparable to the value of 30.5 µM measured for the untagged\nconstruct. Furthermore, after normalization to surface expression levels, the maximal\nvelocities (V ) were also similar between the two variants (Extended Data Fig. 9a).\nmax\nThese new data confirm that the N-terminal GFP-MBP tag does not significantly alter\nthe key kinetic parameters of hTauT. This finding is consistent with a previously\nreported K value of 37.3 µM for a GFP-tagged hTauT construct1. We have\nm\nincorporated these results and their description into the revised manuscript (Lines 143-\n148, Fig. 2a, and Extended Data Fig. 9a).\nWe also sincerely apologize for the typographical error and have corrected \"wide-type\"\nto \"wild-type\" in the legend for Figure 2a.\n2) The number of structures in occluded or inward-open conformation is impressive\nand the authors have made a very good job in deciphering all these. However, since\nstructural biological answers aided by cryo-EM largely relies on assumptions and\ninterpretations of data, it would be helpful if the authors would add data of mutagenesis\nexperiments. The manuscript includes quite a number of very plausible amino acid\ncontact points with substrates or inhibitors; hence, it would be an easy task to mutate\nsome key residues to ascertain loss of function and thereby support the findings of the\ncryo-EM approach (I am especially focusing on Figure 4 for this request).\nResponse: We thank the reviewer for this valuable suggestion and for their positive\nassessment of our structural work. We fully agree that functional validation is crucial\nto corroborate structural observations. In direct response to this comment, we have\nperformed a comprehensive set of mutagenesis experiments targeting key residues\nwithin the substrate-binding pocket identified in our P4S-bound structure (Fig. 4f).\nTo systematically validate these interactions, we designed a series of point mutations\ntargeting residues that coordinate the sulfonate group (e.g., G60, G62, S301), the\nhydrophobic subsite (e.g., L306, L134), and the backbone architecture (e.g., G57,).\nSurface expression assays confirmed that the majority of these mutants were properly\ntrafficked to the membrane, with the notable exception of G62Y, which exhibited\nreduced expression (Revised Extended Data Fig. 11a).\nAs the reviewer anticipated, our functional uptake assays provided strong support for\nour structural model. Mutations of residues that form direct interactions with both P4S\nand the native substrate (e.g., G60Y, L134A, S301A, L306Q) severely impaired or\ncompletely abolished taurine uptake (Fig. 4f). This confirms their essential role in the\nbinding and transport mechanism.\nA particularly insightful result concerns residue E406. Our structure shows that while\nE406 forms a critical salt bridge with the amino group of taurine, it does not interact\ndirectly with P4S, whose amino group is positioned differently due to the rigid\npiperidine ring. Consistent with this precise structural observation, the E406G mutation\nresulted in a complete loss of transport function, underscoring its indispensable role for\nsubstrate binding.\nThese new mutagenesis data, now incorporated in the revised manuscript (Lines 255-\n261, Fig. 4f, and Extended Data Fig. 11a), directly bridge our structural findings with\nfunctional outcomes. They robustly validate the residue-specific interactions we\nidentified and significantly strengthen the biological conclusions of our study.\n3) The question whether cyclic P4S is a substrate or inhibitor cannot be answered by\nthe assay the group has used in this manuscript. This ought to be done in a trans-\nacceleration experiment or also by a release assay: here. tritiated taurine is being\ntransported into the TauT-expressing cells, and then, unlabeled taurine or unlabeled P4S\nis being used in increasing concentrations. In case of taurine, the tritiated taurine will\nbe released by the transporter upon the inward-transport by the taurine applied from the\noutside. However, the problem is that the efflux can be elevated but still not be\ndecisively showing that the compound is a substrate because in some instances,\npseudo-efflux may take place. Since TauT is a member of the SLC6-family and related to\nGABA-transporter, it is dependent on sodium. To determine the substrate status of P4S,\nthe authors should use monensin, a sodium-proton ionophor, which will selectively\nelevate sodium ions intracellularly while depleting protons. This activity will then be\ndecisive to unravel the substrate status of the compound: Only substrates will be\nenhanced in their efflux behavior while inhibitors will not show enhancement of efflux.\nThis has been shown not only for members of the SLC6-MAT family but also for the\nGABA transporter GAT1. The authors shall follow these examples to clearly interrogate\nthe substrate status of P4S.\nWith these additional experiments, the already interesting and broad study will be lend\nsupport – and be even more powerful.\nResponse: We are grateful to the reviewer for this critical suggestion and for providing\nthe definitive methodological framework to resolve the substrate status of P4S.\nFollowing the reviewer's guidance, we have now performed the decisive monensin-\nbased efflux assay.\nAs shown in Revised Fig. 7a, we preloaded HEK293-hTauT cells with [³H]taurine and\nmeasured efflux induced by either unlabeled taurine or P4S. Crucially, pretreatment\nwith monensin—which selectively elevates intracellular sodium levels—significantly\npotentiated the efflux induced by P4S.\nThis Na⁺-dependent potentiation is a definitive hallmark of a translocated substrate, as\nit reports on a Na⁺-coupled exchange process. This result unambiguously distinguishes\na true transported substrate from a static, orthosteric inhibitor, which would not show\nenhanced efflux under these conditions2,3. The data therefore provide conclusive\nevidence that P4S is not merely a competitive inhibitor but a translocatable substrate\nanalog that undergoes the full Na⁺-coupled transport cycle.\nWe have incorporated these findings and their interpretation into the revised manuscript\n(lines 371-376, Fig. 7a). This experiment, as the reviewer predicted, has greatly\nstrengthened our study by providing a powerful functional validation of our structural\nobservations.\nReviewer #2 (Remarks to the Author):\nThe manuscript authored by Jing-Xiang Wu and colleagues reports the structures of\nTauT at a good resolution through cryoEM in complex with taurine, P4S, beta-ala and\nsubstrate free states in different conformations. While the study is technically sound, I\nhave the following major concerns that affect the paper’s quality and raises questions\nthat need to be answered.\nResponse: We appreciate the reviewer's thorough assessment and constructive\ncriticisms, which have significantly strengthened our study.\nThe overall structures of TauT appear to be of a good quality however the authors stop\nat just reporting the TauT interactions and conformational transitions that are generally\nin accordance with the other SLC6 members. There seems to be no attempt to study the\nmolecule in depth by making mutations in and around the binding pocket and observing\ntransport kinetics. There seems to be no particularly insightful observations for the\nmechanism and if there are any characteristics different from other SLC6 members. The\nconclusions are generally weak and really give no added insight into this transporter\ngroup.\nResponse: We thank the reviewer for this critical assessment, which prompted us to\nstrengthen the mechanistic conclusions of our study. In direct response, we have\nperformed extensive mutagenesis of the substrate-binding pocket, and this new\nfunctional data—integrated with our structural findings—has revealed key mechanistic\ninsights that distinguish hTauT from other SLC6 members.\nAs detailed in our revised manuscript (Fig. 4f, Extended Data Fig. 11a, Lines 255-261),\nwe designed and characterized a series of point mutations. Surface expression assays\nconfirmed proper trafficking for most mutants, and functional uptake assays validated\nthe critical role of residues identified in our structures.\nCrucially, this combined approach yielded several novel observations:\nValidation of a Divergent Binding Motif: Our structures reveal that hTauT employs\nan inverted binding architecture compared to monoamine transporters (e.g., hNET).\nIn hTauT, the sulfonate group is coordinated in subsite AL by a cluster of backbone amides\nand serine/hydroxyl side chains (G60, G62, S301), while the amino group is anchored\nin subsite C by a large acidic residue (E406). This is the inverse of the monoamine\ntransporters, where the amino group is in subsite A and the bulky hydrophobic group\npenetrates a larger subsite B. Our mutagenesis confirms that residues defining this\nunique motif—including G60, G62, S301, and E406—are absolutely essential for\ntransport, as the G60Y, G62Y, S301A, and E406G mutants completely abrogate uptake.\nMechanism of Substrate Analog Recognition: The structures of β-alanine-bound and\nP4S-bound hTauT provide unique insights into ligand selectivity. For β-alanine, the\nsubtle but significant reduction in polar interactions for its carboxylate group (with G60\nand S402) offers a clear structural rationale for its slightly lower binding affinity\ncompared to taurine. For P4S, the rigid piperidine ring disrupts the key salt bridge with\nE406 that is essential for taurine coordination. These observations provide a direct\nstructural and mechanistic explanation for the affinities of these ligands, explaining\nwhy β-alanine (Kᵢ = 46.7 μM) and P4S (Kᵢ = 1.4 mM) are lower-affinity substrates or\ninhibitors compared to taurine—a nuance not previously understood.\nLigand-Induced Conformational Selection: A major finding of our study is that P4S\nbinding inverts the intrinsic conformational equilibrium of the transporter. While the\napo state preferentially populates the inward-open conformation (~75%), P4S binding\nselectively stabilizes the inward-occluded state (~75%), a phenomenon we directly\ndemonstrate through particle distribution analysis. This provides a dynamic, energetics-\nbased view of how a ligand can bias the transport cycle, moving beyond a static\nstructural description.\nIn summary, the new mutagenesis data rigorously validate our structural models and\nallow us to advance from observation to mechanistic understanding. Our work now\nestablishes the unique, inverted binding motif of hTauT, explains the structural basis\nfor substrate analog affinity, and reveals a principle of ligand-induced conformational\nselection. We believe these findings provide significant new insight into the SLC6\nfamily by elucidating key structural determinants of substrate specificity and the\ndynamic regulation of the transport cycle.\nFurther the density of P4S seems to be weak in the occluded state of the transporter.\nThe authors need to give a contoured density separately of P4S. Have any binding sites\nof P4S observed in the inward-open state of the transporter since the structure in inward\nopen state says that it is P4S inward in fig 1e. A more detailed, direct biochemical\nevidence is required to ascertain if P4S is indeed a transporter. It cannot be based on\nstructural comparisons.\nResponse: We thank the reviewer for these critical and constructive suggestions, which\nhave been instrumental in strengthening the evidence for our conclusions. We have\naddressed these concerns with substantial new experimental data, including improved\nstructural maps and a definitive functional assay.\nAddressing the Density and Map Quality for P4S: We agree that clear ligand density\nis paramount. To resolve this, we collected additional cryo-EM data. This effort\nimproved the resolution of our P4S-bound structures to 2.8 Å (inward-occluded)\nand 3.1 Å (inward-open), resulting in clear and unambiguous density for the P4S molecule\nin both conformational states (Revised Extended Data Fig. 5). As requested, we now\nprovide the contoured density for P4S separately in the revised figures (Revised Fig. 4c\nand 5b), allowing for direct assessment of its placement.\nConfirming P4S Binding in the Inward-Open State: The reviewer correctly inquired\nabout P4S binding in the inward-open state. Our improved structure confirms that a\nwell-defined density for P4S is indeed present in the substrate-binding site of the\ninward-open conformation, a feature absent in the apo state (Fig. 5b). The ligand's\nconfiguration is nearly identical between the two states. In the inward-open\nconformation, the sulfonate group remains engaged with subsite AL, forming hydrogen\nbonds with G62 (TM1), Y138 (TM3), S301 (TM6), and S402 (TM8) (Fig. 5c).\nThe principal conformational change is an ~47° outward splaying of TM1a, which\nsevers the hydrogen bond with G60 and disrupts the Na1 site, thereby partially\ndestabilizing P4S interactions (Fig. 5d). This mechanism of ligand retention in a\npartially weakened state during a gating transition, which parallels observations in\nnoradrenaline-bound hNET structures4. These data confirm that P4S remains bound\nthroughout the conformational cycle.\nProviding Direct Biochemical Evidence that P4S is a Transportable Substrate: As\nthe reviewer rightly emphasizes, structural comparisons alone are insufficient to prove\ntransportability. Therefore, we performed the decisive monensin-based efflux assay, a\ngold-standard functional test for SLC6 transporters. In HEK293-hTauT cells preloaded\nwith [³H]taurine, P4S induced robust efflux, similar to the native substrate taurine.\nCrucially, pretreatment with monensin—which elevates intracellular [Na⁺]—\nsignificantly augmented the P4S-induced efflux (Fig. 7a). This Na⁺-dependent\npotentiation is a hallmark of a true transported substrate engaging in exchange transport\nand unambiguously distinguishes it from a static inhibitor. Therefore, we conclude with\nhigh confidence that P4S is a translocatable substrate analog, not merely an orthosteric\ninhibitor.\nThese new structural and functional data have been incorporated into the revised\nmanuscript (Fig. 4c, 5b-d, 7a, and associated text). We believe these additions provide\na conclusive and multi-faceted validation of our model.\nThe Authors do not seem to report the presence of any bound detergents or lipids despite\nachieving a high resolution. In most cases some cholesterol molecules tend to be visible\nwhen DDM CHS is used for structure determination.\nResponse: We thank the reviewer for this insightful suggestion regarding the potential\npresence of bound lipids or detergents. In response, we conducted a more detailed\nanalysis of our cryo-EM maps, particularly following further data collection and\nprocessing that improved the overall resolution, with the β-alanine-bound hTauT\nstructure now reaching 2.7 Å.\nAs the reviewer anticipated, the enhanced quality of our maps revealed clear, non-\nprotein densities at conserved sites known to bind cholesterol or CHS (cholesterol\nhemisuccinate) in other SLC6 family transporters. We have successfully modeled these\ncholesterol/CHS molecules into their respective densities.\nA new panel, Extended Data Fig. 8, has been added to the revised manuscript to\nexplicitly show the location and density fit of these bound molecules. The\naccompanying text in lines 134-141 describes their positions and potential functional\nroles, aligning with the established importance of lipid interactions in the stability and\nfunction of SLC6 transporters.\nWe believe this addition, made possible by our high-resolution structures, provides a\nmore complete structural description of hTauT and its membrane environment.\nbeta-alanine substrate competition and uptake could have been studied to provide more\ninsights into the ability to compete with taurine. This was only done as a qualitative\nassay whereas a K measurement could have been done with beta-alanine.\ni\nResponse: We thank the reviewer for this excellent suggestion. In direct response, we\nhave now quantified the inhibitory effect of β-alanine on hTauT-mediated taurine\nuptake.\nWe performed a dose-response assay, which demonstrates that β-alanine acts as a potent,\ndose-dependent competitive inhibitor of taurine uptake (Revised Fig. 3a). The data\nyield an IC₅₀ value of 48.96 μM, from which we calculated a Kᵢ value of 46.7 μM. This\nmeasured affinity aligns remarkably well with the previously reported value of 44.5\nμM1, thereby validating our experimental approach.\nThese new quantitative data provide crucial kinetic parameters that strengthen our\nstructural observations and offer deeper insight into the competitive binding dynamics\nbetween these two substrates at the molecular level. The results have been incorporated\ninto the revised manuscript (Lines 214-217, Fig. 3a).\nThe subsite organization demonstrated here does not highlight altered organization\nobserved in GATs in comparison to the MATs reported earlier. The proper literature for\nthis subsite organization has to be cited as well.\nResponse: We thank the reviewer for this insightful suggestion regarding the\ncomparative subsite organization. We have now substantially expanded our analysis to\nhighlight the distinctive subsite organization of hTauT and related GABA transporters\n(GATs) compared to monoamine transporters (MATs), citing the foundational literature\nthroughout the revised manuscript.\nSpecifically, we have added a detailed comparative analysis section that explicitly\ndescribes how the substrate-binding architecture in hTauT and GATs represents an\ninverted configuration relative to MATs. As noted in the rGAT1 structure, \"The primary\nbinding site of biogenic amine transporters is represented as a group of subsites (A,\nB\nand C)... In the GAT1 binding site environment, the trilobed binding site is altered to a\nbean-shaped binding site with subsites A and C'\"5. Similarly, the hGAT1 study observes\nthat \"The substrate binding modes of biogenic amine NSS members such as SERT and\ndDAT are quite different from that of GAT1\"6.\nOur analysis builds upon these foundational observations to demonstrate that hTauT\nshares this altered organization with GATs. The key distinction lies in the inversion of\ncritical residues: in MATs like hNET, a large acidic residue (D75) anchors the\nsubstrate's amino group in subsite A, whereas in hTauT, this position is occupied by a\nsmall glycine (G60) that accommodates the sulfonate group in an enlarged subsite AL.\nConversely, the position occupied by a glycine in MATs contains a large acidic residue\n(E406) in hTauT that coordinates the amino group in subsite C.\nThis comparative analysis, supported by the appropriate citations to hGAT16 and\nrGAT15 structures, has been incorporated throughout the revised manuscript (Lines\n160-167, Extended Data Fig. 10) and provides crucial context for understanding the\nevolutionary adaptation of the SLC6 transporter scaffold.\nOverall the study study reports a set of TauT structures but the conclusions that could\nhave been made through a detailed analyses of substrate recognition and transport\nmechanisms are lacking.\nResponse: We thank the reviewer for this critical assessment, which prompted us to\nsignificantly strengthen the analytical depth and mechanistic conclusions of our study.\nWe have completely rewritten and expanded the conclusion section to move beyond a\ndescriptive summary of structures (Lines 394-431). The new text now provides a\nsynthesized, mechanism-focused narrative that integrates our structural, kinetic, and\nmutagenesis data to deliver clear insights into substrate recognition and the transport\ncycle.\nThe core of this new analysis is presented below. It now explicitly:\nArticulates a Mechanism for Substrate Specificity: We define the unique, inverted\nbinding motif of hTauT and explain how specific residues (e.g., the glycine-rich\nmotif, E406) structurally determine affinity for taurine versus β-alanine and P4S.\nProposes a Dynamic Transport Model: We synthesize observations from multiple\nstructures into a coherent, stepwise model for intracellular gating and ion/substrate\nrelease, identifying TM1a as the key conformational lever.\nReclassifies a Classic Inhibitor: We use our integrated data to resolve the\nmechanism of P4S action, conclusively identifying it as a transported substrate\nanalog.\nWe are confident that these substantive additions directly address your concern and\nelevate the manuscript's significance by providing the detailed mechanistic analysis\nthat was previously lacking.\nSome more minor comments are given below.\n- Was the thermal stability assay done using the protein containing GFP-MBP? The\nhigher melting point can be due to the presence of GFP and MBP tags.\nResponse: We thank the reviewer for raising this critical point regarding the potential\ninfluence of the fusion tags on thermal stability.\nThe reviewer is correct to highlight this. Our initial thermal stability screening was\nconducted using unpurified, GFP-MBP-tagged hTauT in cell lysates via FSEC.\nTo definitively determine the tag's contribution, we subsequently purified both the GFP-\nMBP-tagged and untagged hTauT proteins and performed a quantitative comparison.\nThe data confirm that the GFP-MBP tag itself confers a significant stabilization, with\nthe tagged construct exhibiting a melting temperature (T ) of 82 °C, compared to 46 °C\nm\nfor the untagged protein (Extended Data Fig. 1a).\nWe have incorporated these new data and a clarified description of the methodology\ninto the revised manuscript (Lines 441-451 and 475-483) to ensure this distinction is\nunambiguous for the reader.\n- Expression construct has no mention of strep tag but in purification, streptactin beads\nwere used for affinity chromatography.\nResponse: We thank the reviewer for their careful attention to this detail. The construct\nindeed contains a dual Strep-tag II affinity tag, which was engineered into a solvent-\nexposed loop of the GFP moiety. This tag was utilized for affinity purification on\nstreptactin resin.\nWe apologize for this omission in the original manuscript and have now explicitly\ndetailed the presence and location of the Strep-tag in the Methods section (Lines 443-\n444) of the revised version.\n- In native PAGE, there is a band which corresponds to MW between 130 to 180 kDa.\nCould it be some oligomeric species?\nResponse: We thank the reviewer for this insightful question regarding potential\noligomeric species.\nThe band observed between 130-180 kDa in the original SDS-PAGE (Extended Data\nFig. 1b, middle) corresponds to the monomeric GFP-MBP-hTauT fusion protein, which\nhas a calculated molecular weight of approximately 144 kDa.\nTo directly and unequivocally address the question of oligomerization in the native state,\nwe have now performed native-PAGE analysis on the SEC peak fractions. As shown in\nthe new Revised Extended Data Fig. 1b, right, the results display a single, dominant\nband, confirming the monodisperse nature of the sample and the absence of significant\noligomeric species.\nWe confirm that these monodisperse fractions were used for cryo-EM grid preparation.\nThis new data and its interpretation have been incorporated into the revised manuscript\n(Lines 88-92 and Revised Extended Data Fig. 1b).\nReviewer #3 (Remarks to the Author):\nThe manuscript “Structural mechanism of substrate binding and inhibition of human\ntaurine transporter” describes 6 cryo-EM structures of human taurine transporter (TauT,\nSLC6A6) bound to taurine, b-alanine, competitive inhibitor P4S, and in substrate-free\nstates. This work is the first presentation of the therapeutically important target, the\nhuman TauT structures. While the cryo-EM density maps are of good quality, and the\nstudy provides interesting details of the transport cycle, the manuscript can benefit from\nmore in-depth discussion of the findings, placing the study in the context of the wealth\nof structural and functional data available on SLC6 transporters.\nResponse: We thank the reviewer for their positive feedback and for the detailed\nsuggestions that have greatly improved the depth and context of our manuscript.\nNotable issues:\n1. There is no mention of TM5 unwinding and solvation in inward-open states, a\ncharacteristic of inward facing states of SLC6 transporters. Whether or not this can be\nobserved, should be discussed. See the reference: Malinauskaite L, et al., doi:\n10.1038/nsmb.2894. Epub 2014 Oct 5. PMID: 25282149\nResponse: We thank the reviewer for this critical observation and for directing us to the\nfoundational reference by Malinauskaite et al. We have now incorporated a detailed\nanalysis of TM5 conformational changes in our inward-open states as suggested.\nConsistent with the characteristic inward-facing state of SLC6 transporters, our\nstructures of both the apo and P4S-bound hTauT reveal a partially unwound\nintracellular segment of TM5 (Revised Fig. 5a and Extended Data Fig. 11b). As\nhighlighted in the referenced work, this unwinding is a key mechanistic feature that\nfacilitates the solvation of the intracellular vestibule and the Na2 site, priming the\ntransporter for substrate and ion release.\nA comparative analysis shows that the TM5 conformation in hTauT closely aligns with\nthe inward-open states of human transporters hGAT1, hGAT3, and hGlyT1, but is\ndistinct from the outward-tilted TM5 helix observed in the bacterial homolog LeuT\n(Revised Extended Data Fig. 11c).\nThis analysis and the appropriate citation have been incorporated into the revised\nmanuscript (Lines 264-271) to provide proper mechanistic context for our structural\nfindings.\n2. The inward facing states presented in this study, are “inward-open” states, please\ncorrect the text accordingly.\nResponse: We thank the reviewer for your suggestions. We have corrected this\nexpression throughout the revised manuscript.\n3. Please elaborate on the occluded states, are they outward/inward occluded, or how\ndo they compare to previous SLC6 occluded states, the most similar ones? 249 – 259\ndiscusses this part to some extent but requires further elaboration.\nResponse: We thank the reviewer for prompting this important clarification. We have\nsignificantly elaborated on the nature of the occluded states in the revised manuscript.\nAll our substrate/inhibitor-bound occluded structures (taurine, β-alanine, and P4S)\nare definitively in the inward-occluded conformation. This classification is based on a key\nstructural hallmark: the unwinding of the first turn on the intracellular side of TM5 at\nthe conserved Gly(X9)Pro motif (G243(X9)P253 in hTauT), which leads to hydration\nof the Na2 site (Figs. 2b, 3b, 4b). This feature is characteristic of inward-occluded states\nin SLC6 transporters, as established in structures of hGAT1 and MhsT (Extended Data\nFig. 9b).\nTo provide further context, we performed a structural comparison with other SLC6\ntransporters in similar states. Our analysis confirms that the inward-occluded\nconformation of hTauT is most similar to that of GABA-bound hGAT1, with a Cα\nRMSD of 1.627 Å (Extended Data Fig. 10a). This quantitative comparison solidifies\nour conformational assignment and integrates our findings within the existing structural\nlandscape of the SLC6 family.\nThese clarifications and data have been incorporated into lines 155-160 and Fig. 2b, 3b,\n4b, and Extended Data Fig. 9b in the revised manuscript.\n4. Line 265: ‘and are alternative substrates’, and line 259: ‘This suggests that P4S might\nbe an alternative substrate to hTauT’. This is too definitive based on the evidence\npresented in the manuscript as to whether P4S is an alternative substrate or substrate\nanalogue. It has only been speculated from structural comparisons, but no functional\nexperiments were carried out. Therefore, it would be best to be more speculative with\nthis statement.\nResponse: We thank the reviewer for this critical point and agree that functional\nvalidation was essential. In response, we have conducted the decisive monensin-\nenhanced efflux assay to definitively determine the transport status of P4S.\nOur new functional data demonstrate that P4S induces efflux of preloaded [³H]taurine\nfrom HEK293-hTauT cells (Revised Fig. 7a). Crucially, pretreatment with monensin -\nwhich elevates intracellular sodium concentration - significantly potentiated this P4S-\ninduced efflux, mirroring the effect observed with the native substrate taurine (Lines\n371-376 and Revised Fig. 7a). This monensin-dependent potentiation is a well-\nestablished characteristic of transported substrates in SLC6 transporters, as it reflects\nsodium-coupled exchange2,3.\nBased on these comprehensive structural and functional results, we now conclusively\nstate that P4S acts as a translocated substrate of hTauT, competitively inhibiting taurine\nuptake by undergoing the full transport cycle. We have revised the relevant statements\nin the manuscript accordingly and included these new data (Fig. 7a and corresponding\ntext).\n5. Binding site comparison of taurine-bound TauT with PAT1 (SLC36A1), another\ntaurine transporter, on sequence level (and perhaps on a model basis – alphafold model)\ncould be relevant.\nResponse: We thank the reviewer for this excellent suggestion. To provide a deeper\nevolutionary and mechanistic context, we have performed a detailed comparative\nanalysis of the substrate-binding sites of hTauT (SLC6A6) and hPAT1 (SLC36A1).\nOur analysis, which combines sequence alignment with a structural comparison using\nan AlphaFold3-predicted model of hPAT1, reveals a mixed picture of conservation and\ndivergence. We find that key structural features enabling sulfonate group recognition\nare conserved: the small residues that form the enlarged subsite AL in hTauT (G57,\nG60, G62, S301) are preserved in hPAT1 (G59, G62, G64, S268). Furthermore, residues\ncoordinating the substrate's amino group are conservatively substituted (F300 and E406\nin hTauT correspond to F267 and Q350 in hPAT1).\nHowever, a critical difference was identified at a position crucial for sodium\ncoordination. The unique F58 in hTauT, which helps coordinate the Na+ ion at the Na1\nsite, is not conserved in hPAT1. This divergence in the precise architecture of the\nsodium-binding site likely underlies the distinct transport properties of the two\ntransporters, contributing to hPAT1's characteristically low affinity for taurine (K > 1\nm\nmM) and broader substrate promiscuity, in contrast to the high-affinity, selective\ntransport by hTauT.\nThis new comparative analysis has been incorporated into the revised manuscript (Lines\n180-192, Extended Data Fig. 9c, d) and provides valuable insight into how different\ntransporter families have evolved distinct solutions for recognizing the same substrate.\n6. The order of figures does not follow the order they are mentioned in the text, please\ncorrect that.\nResponse: We sincerely apologize for this oversight. We have now thoroughly checked\nthe entire manuscript and corrected the figure order to ensure it corresponds precisely\nwith the order of mention in the text.\n7. Uptake assay: It is not clear how many replicates were conducted in this assay and\nwhat data is being shown (mean? Single data point?).\nResponse: We thank the reviewer for highlighting this lack of clarity. All uptake\nassays were performed with three independent biological replicates. The data points in\nthe figures represent the mean values, and the error bars indicate the standard error of\nthe mean (S.E.M.). We have explicitly stated this information in the respective figure\nlegends of the revised manuscript.\n8. Is there any indication that P4S gets transported by TauT, whether based on previous\nstudies or experiments performed by the authors?\nResponse: We thank the reviewer for this important question regarding the transport\nstatus of P4S. Our conclusion that P4S is a transported substrate is supported by\nmultiple lines of evidence:\nFirst, our structural data demonstrate that P4S binds to both inward-occluded and\ninward-open conformations of hTauT, similar to how native substrates like\nnoradrenaline bind to multiple conformational states in related transporters hNET4.\nStructural analysis further confirms that P4S binding is compatible with all major\nconformational states in the transport cycle, identical to what we observe for\nphysiological substrates taurine and β-alanine (Revised Fig. 7b).\nSecond, previous literature has characterized P4S as both a substrate analog and\ncompetitive inhibitor of hTauT1.\nMost definitively, we conducted a decisive monensin-enhanced efflux assay to directly\ntest the substrate properties of P4S. In HEK293-hTauT cells preloaded with [³H]taurine,\nP4S induced efflux comparable to unlabeled taurine. Crucially, monensin pretreatment\n- which elevates intracellular sodium concentration - significantly potentiated P4S-\ninduced efflux, mirroring the effect observed with native taurine. This monensin-\ndependent potentiation is a well-established characteristic specific to transported\nsubstrates in SLC6 transporters, as it reflects sodium-coupled exchange kinetics (Lines\n371-376 and Fig. 7a).\nTherefore, based on comprehensive structural compatibility, previous literature reports,\nand definitive functional evidence from the monensin-enhanced efflux assay, we\nconclude that P4S acts as a translocatable substrate analog that undergoes the full\ntransport cycle while competitively inhibiting taurine uptake.\n9. Local density figure, currently extended data figure 6, should be provided for all the\nmodels and maps presented in the manuscript, separately, and not just representative\nsections of selected models and maps.\nResponse: We thank the reviewer for this valuable suggestion. In accordance with this\nrecommendation, we have now generated and provided local density for all structural\nmodels determined in this study. These are presented in the revised Extended Data\nFigure 6, which separately displays the density for each key structural element. This\nprovides full transparency and allows for direct assessment of map quality and model\ninterpretation throughout our structural dataset.\n10. Distances should be mentioned for the presented interactions on figures.\nResponse: We thank the reviewer for this suggestion. We have now measured and\nlabeled the key interaction distances (e.g., for hydrogen bonds and salt bridges) in all\nrelevant figures throughout the manuscript.\n11. Label the TMs on figures, please.\nResponse: We thank the reviewer for this suggestion. We have now labeled all TMs in\nthe relevant structural figures throughout the manuscript to improve clarity and\norientation for readers.\n12. The sigma levels are correctly reported for all the presented density figures;\nhowever, it is not clear if they are in reference to Chimera, coot, or? please specify that\nin the figure legends.\nResponse: We thank the reviewer for highlighting the need for this clarification. The\nsigma (σ) contour levels reported for all cryo-EM density maps in our figures were\ndetermined using UCSF ChimeraX. We have updated all relevant figure legends in the\nrevised manuscript to explicitly state that map contours are displayed at the indicated\nsigma levels as calculated in ChimeraX.\n13. Line 250: The outward-open structure of GlyT1 is not substrate-free! It is bound to\ncompetitive inhibitor PF03463275.\nResponse: We thank the reviewer for catching this important inaccuracy. The reviewer\nis absolutely correct. We have now corrected the manuscript to accurately state that the\noutward-open hGlyT1 structure used for comparison is bound to the competitive\ninhibitor PF-03463275, not substrate-free. The revised text in line 380 has been\namended accordingly.\nModels and maps:\n1. There is no density for NAG sites, e.g., in the map 2638, please remove the NAGs\nfrom this model and others if the density is not supporting the modelling.\nResponse: We thank the reviewer for this careful observation. We have now removed\nall NAG moieties from our structural models at positions N163, N179, and N190, as\nthe corresponding cryo-EM density lacks sufficient detail to support their modeling.\nThe models have been updated accordingly.\n2. P4S-bound model – occluded state:\nThe fit of the inhibitor could be optimised, the good quality of the density should allow\na slightly better fitting. There appears to an interaction of nitrogen group of P4S with\nCO of E409, if the pose of P4S is modified to fit the density better. If that is the case,\nthe pose should be optimised to see if the interaction is possible.\nResponse: We thank the reviewer for this detailed structural feedback. We have\noptimized the fitting of the P4S molecule into the improved 2.8 Å resolution cryo-EM\ndensity map of the inward-occluded state. The current model shows excellent\nagreement with the experimental density (Extended Data Fig. 6). We observe an\ninteraction of nitrogen group of P4S with backbone carbonyl oxygen (CO) of F300.\nHowever, regarding the potential interaction with residue G409, we confirm that the\nnitrogen group of P4S is positioned at a distance greater than 4.0 Å from the carbonyl\noxygen of G409, which is beyond the range for a meaningful hydrogen bond interaction.\nThe current refined pose therefore represents the optimal fit to the electron density\nwhile maintaining proper stereochemistry.\n3. P4S-bound model – presented as the inward-open\nIt is quite important to clarify whether the P4S also stabilises an inward-open state, or\nthe density in binding pocket accommodates another small molecule. The general fit of\nthe model into density is not finalised, the map is noisy. The noise could originate from\nthe local refinement, if a mask has been used, and needs to be optimised/improved to\nbe evaluated.\nResponse: We thank the reviewer for these critical questions regarding the P4S-bound\ninward-open conformation. We have addressed these concerns through substantial\nimprovements to our structural data and analysis.\nWe collected additional cryo-EM data, improving the resolution of the P4S-bound\nstructures to 2.8 Å (inward-occluded) and 3.1 Å (inward-open), which provides\nunambiguous density for modeling P4S in both conformational states (Extended Data\nFig. 5). The ligand maintains nearly identical configuration between these states (Fig.\n5d), with its sulfonate group forming conserved hydrogen bonds with G62 (TM1),\nY138 (TM3), S301 (TM6), and S402 (TM8) (Figs. 4d, 5c).\nThe key conformational change involves ~47° outward splaying of TM1a in the inward-\nopen state, which disrupts specific P4S interactions including the hydrogen bond with\nG60 and Na⁺-mediated coordination with F58 and N63 (Figs. 5c, d). This pattern of\npreserved binding mode with partially destabilized interactions during gating\ntransitions mirrors observations in noradrenaline-bound hNET structures4, where\nsimilar TM1a displacement (F72 in hNET) weakens substrate contacts in the inward-\nopen state (Extended Data Fig. 11d).\nSubstrate-free states:\n1. The substrate-free states can benefit from a section in the manuscript, clearly\ndescribing the two states, discussion on differences, if and how they were obtained.\nThey are obtained from one data set, and it’s quite amazing considering the data set is\nnot very large. Please elaborate on the two states, the how, the why, and descriptions in\na separate section, similar to other states.\nResponse: We thank the reviewer for this excellent suggestion, which has significantly\nstrengthened our manuscript. In response, we have now created a dedicated section\nentitled \"Conformational equilibrium of apo and P4S-bound hTauT\" to provide a\ncomprehensive description of the substrate-free states, mirroring the detailed analysis\nprovided for other states in the paper.\nWe have elaborated on the methodology in both the Results and Methods sections.\nSpecifically, we detail how the inward-occluded and inward-open apo states were\nresolved from a single cryo-EM dataset. This separation was achieved through\nextensive 3D variability analysis (3DVA) and focused 3D classification, which\nsuccessfully deconvoluted the pronounced conformational heterogeneity, particularly\nwithin the dynamic TM1a helix. We agree with the reviewer that resolving two distinct\nand well-defined conformational states from one dataset is a notable technical outcome\nthat underscores the dynamic nature of the transporter.\nThe following paragraph, now included in the manuscript (Lines 92-106), describes this\nmethodological approach:\n“Initial single-particle processing of the apo and P4S-bound datasets indicated\nconformational heterogeneity, particularly in the TM1a helix, as evidenced by its\ndiscontinuous and poorly resolved density. This suggested significant flexibility,\ncontrasting with the well-defined TM1a density observed in the stable, substrate-bound\n(taurine/β-alanine) states. To resolve this heterogeneity, we employed 3D variability\nanalysis (3DVA) in cryoSPARC, which directly visualized the substantial flexibility of\nTM1a (Extended Data Movies 1-2). Subsequent focused 3D classification and\nrefinement successfully separated two distinct conformations from these datasets:\ninward-occluded and inward-open (Extended Data Figs. 2 and 5). This strategy enabled\nus to determine high-resolution structures of hTauT in multiple conformational states:\nthe apo state in the inward-occluded conformation and the inward-open conformation\n(2.8 Å and 3.1 Å, respectively), the taurine-bound state in the inward-occluded\nconformation at 2.9 Å, the β-alanine-bound state in the inward-occluded conformation\nat 2.7 Å, and the P4S-bound state in the inward-occluded conformation and inward-\nopen conformation (2.8 Å and 3.1 Å, respectively) (Extended Data Figs. 2-5 and\nExtended Data Table 1).”\nFurthermore, in the new dedicated section, we provide a clear description of the two\nsubstrate-free states, their structural features, and the key differences between them.\nThe section highlights that the transition from the inward-occluded to the inward-open\nstate is primarily governed by a ~38° outward splaying of TM1a and further unwinding\nof TM5, which opens a cytoplasmic pathway. This analysis provides a clear structural\nview of the conformational equilibrium in the absence of substrate and directly leads to\nour model of a dynamic transporter ensemble.\nThe core of this new section is presented below (Lines 297-318):\n“Following 3DVA and focused 3D classification, we resolved two distinct inward\nconformations of apo hTauT (Fig. 1a, b). The inward-occluded conformation,\nreconstructed from 149,606 particles, features an occluded substrate-binding site and a\npartially unwound intracellular segment of TM5 (Extended Data Fig. 12a). This\nconformation is nearly identical to the taurine-bound inward-occluded state, with a\nRMSD of 1.250 Å over 550 aligned Cα atoms (Extended Data Fig. 12b). In contrast,\nthe inward-open conformation, determined from 322,377 particles (~75%), is virtually\nsuperimposable with the P4S-bound inward-open structure (RMSD 0.372 Å; Extended\nData Fig. 11b). A comparison between the two apo conformations reveals that the\ntransition to the inward-open conformation is characterized by a ~38° outward splaying\nof TM1a and further unwinding of the intracellular side of TM5, which opens a\ncytoplasmic pathway (Fig. 6a). The predominance of the inward-open conformation in\nthe apo state indicates that apo hTauT preferentially populates this state. The\ncoexistence of both conformations suggests the apo transporter samples a dynamic\nconformational ensemble, which likely includes outward conformations. We therefore\npropose that apo hTauT exists in a dynamic equilibrium; when it transiently visits an\noutward-open conformation, taurine binding primes the transporter for translocation.\nThis intrinsic conformational plasticity is a shared feature among SLC6 transporters,\nsuch as GlyT1 and SERT7,8.\nStrikingly, P4S binding inverts this equilibrium. In the P4S-bound state, the majority of\nparticles (~75%) populate the inward-occluded conformation, with a minority (~25%)\nin the inward-open state. This pronounced population shift demonstrates that P4S\nbinding preferably stabilizes the inward-occluded conformation, which is structurally\nrationalized by the enhanced ligand-protein interactions observed in this state (Fig. 5d).”\n2. In map 2630, there is density around the TM5 region that is not modelled. Could the\nauthors comment on the density whether it is noise (that should be processed better) or\nif the TM5 can be partially modelled into that density? The map is still noisy, and the\nmodel fit at the turns/loop regions can clearly be improved.\nResponse: We thank the reviewer for this critical observation regarding the quality of\nour initial cryo-EM map 2630. In direct response, we performed optimized image\nprocessing. This has resulted in a significantly improved global resolution from\nprevious 3.65 Å to 3.12 Å for our map (Revised Extended Data Fig. 5).\nIn the revised, higher-resolution reconstruction, the density in the TM5 region is now\nclear, allowing for modeling of this helix, including its connecting loops. The improved\nmap quality throughout the structure has enabled better model fitting in the flexible\nregions. The updated model and corresponding map are now presented.\n3. The disulfide bridge C162-C171 in EL2 seems to be broken in map 2638, please\ncomment on this and other maps.\nResponse: We thank the reviewer for this careful observation. We have now improved\nthe resolution and quality of all our cryo-EM maps through optimized data processing.\nIn these revised maps, the disulfide bridge between C162 and C171 in the EL2 loop is\nclearly defined and intact across all structural models, including the one corresponding\nto the previously noted map (2638). We have included representative density for this\ndisulfide bond in the revised figures (Extended Data Fig. 6) to confirm its presence and\nproper geometry in all determined structures.\nβ-alanine-bound state:\nThere is extra density in the binding pocket. It could nicely accommodate a water\nmolecule and a water mediated interaction of b-alanine with E403.\nResponse: We thank the reviewer for this excellent suggestion. We have modeled a\nwater molecule into the corresponding extra density within the substrate-binding pocket\nof the β-alanine-bound structure. This water molecule is now included in our refined\nmodel and is shown to mediate an interaction between the amino group of β-alanine\nand the side chain of Q403 on TM8, as visualized in the revised Figure 3d.\nPlease consider modelling the Na1 in the b-alanine-bound state, the density is not as\nstrong, but resealable.\nResponse: We thank the reviewer for this suggestion. We have modeled the Na+ ion at\nthe Na1 site in the β-alanine-bound structure.\nTaurine-bound state\nExtra density in the binding pocket of Taurine-bound state could be again coordinating\nwater molecule with the Nitrogen of the substrate.\nResponse: We thank the reviewer for this insightful suggestion. We have modeled a\nwater molecule into the corresponding unassigned density within the substrate-binding\npocket of the taurine-bound structure. This water molecule is now included in our\nrefined model and coordinated with the amino group of taurine, as shown in the revised\nFigure 2d.\nIons binding site\nPlease add the density to ion binding site figures.\nResponse: We thank the reviewer for this suggestion. We have now added the\ncorresponding cryo-EM density to the figures depicting the Na+ and Cl⁻ (revised Figure\n6e-g). The clear density observed at these sites provides direct visual evidence\nsupporting our assignment and modeling of these functionally essential ions.\nFigures\nFigure 1 – Please present all the models and respective maps at least once in the\nmanuscript.\nResponse: We thank the reviewer for this suggestion to improve the clarity of our\nstructural presentation. We confirm that all structural models and their corresponding\ncryo-EM density maps determined in this study have now been explicitly presented in\nthe revised Figure 1. Additionally, each structure is featured in subsequent figures with\nits respective map to allow for detailed examination of specific conformational states\nand ligand-binding interactions. The local map quality for all models is further\ndemonstrated in Extended Data Figure 6.\nChange ‘functional states’ to ‘conformational states’\nResponse: We thank the reviewer for this precise terminology suggestion. We have\nsystematically replaced the term \"functional states\" with \"conformational states\"\nthroughout the manuscript to more accurately describe the structural variations captured\nin our study.\na) Report the density level of the map used for the figure.\ne) hTauT is bound to P4S in two different conformations however P4S is not shown in\nthe inward conformation as it was not fitted. It may be beneficial showing the density\nthat you do observe in this part of the figure (please see above comments as this\nmap/model needs further clarification).\nResponse: We thank the reviewer for these important suggestions regarding figure\npresentation.\na) We have now reported the specific contour levels for all cryo-EM maps in their\nrespective figure legends throughout the manuscript.\ne) We have substantially improved the structural data for the P4S-bound states.\nThrough\nadditional data collection and processing, we achieved improved resolutions of 2.8 Å\n(inward-occluded) and 3.1 Å (inward-open). The enhanced map quality now allows\nunambiguous modeling of P4S in both conformational states. We have created a new\nFigure 5 that specifically focuses on the P4S-bound inward-open conformation, clearly\nshowing both the molecular model and the corresponding electron density for the ligand.\nThis provides direct visualization of P4S binding in this state and facilitates comparison\nwith the inward-occluded conformation.\nFigure 3a\nβ-Alanine binding mode “by” hTauT. Replace “by” with at, or in.\nNext line as well: The inhibition of β-alanine “to” hTauT – also not clear what it means?\nIsn’t β-Alanine a substrate? It is not clear what this graph is showing and why it is\npresented as a bar plot. How many independent experiments were performed? If the\nauthors are showing replacement of hot-taurine with β-Alanine, the data should be\npresented more clearly.\nResponse: We thank the reviewer for these important corrections and suggestions\nregarding terminology and data presentation in Figure 3a.\nWe have corrected the phrasing throughout to properly describe \"β-alanine binding\nmode in hTauT\" and \"inhibition of hTauT by β-alanine.\" As the reviewer noted, β-\nalanine is indeed a known substrate of hTauT, as demonstrated in previous studies1. To\naddress the concern about data presentation, we have replaced the original bar plot with\na concentration-response curve showing inhibition of [³H]taurine uptake by β-alanine.\nThis new presentation clearly demonstrates the dose-dependent competitive inhibition\nand allows determination of the IC₅₀ value (the calculated K = 46.7 µM). The data\ni\nrepresent means ± S.E.M. from three independent experiments, ensuring statistical\nreliability. This revised figure now more effectively communicates the competitive\nrelationship between β-alanine and taurine for hTauT binding and transport.\nFigure 5. Please outline how the degrees of motion were measured. Label all the TMs\nin the individual panels, not just TM1.\nResponse: We thank the reviewer for these important suggestions to improve the clarity\nof Figure 5 (Revised Fig. 6).\nWe have now labeled all relevant TMs in each panel of the revised figure 6 to provide\nbetter structural context. Additionally, we have added a detailed description in the figure\nlegend explaining how the degrees of motion were quantified. Specifically, the\nconformational changes were measured by calculating the angles between equivalent\nCα atoms in the TM1a helix across different states using PyMOL's measurement tools,\nwith the hinge point defined at the conserved glycine residues in the TM1 hinge region.\nThis methodological detail ensures transparency in our structural analysis.\nExtended data figure 1. Outline the detergent concentrations used for the experiments\nconducted in panel a. The screening of detergents was not outlined in the Methods.\nResponse: We thank the reviewer for this professional suggestion to improve the\nmethodological documentation. We have now added the specific detergent\nconcentrations used for the FSEC-based thermostability screening in the legend of\nExtended Data Figure 1a. Additionally, we have expanded the Methods section to\ninclude a detailed description of our detergent screening procedure, including the\nspecific detergents and concentrations tested, as well as the experimental protocol for\nevaluating protein stability under different detergent conditions (Lines 441-451 and\n475-483). These revisions provide complete transparency regarding our optimization\nprocess.\nExtended Data Table 1:\nInitial model used: please provide the respective alphafold model ID(s).\nResponse: We have provided the respective alphafold model ID in the revised Extended\nData Table 1.\nPlease add the CaBLAM values for the models (it should be below 1% to be acceptable).\nResponse: We thank the reviewer for this suggestion regarding model validation. We\nhave now included the CaBLAM outlier scores for all structural models in the revised\nExtended Data Table 1. All models show CaBLAM values below 1%, confirming the\nproper geometry of our protein backbone conformations. These comprehensive\nvalidation statistics are now available in the updated Extended Data Table 1.\nExtended data figure 9, g, the label of the top model is placed behind the figure. The\nfigure can be expanded accommodating the various states identified in this study and\nperhaps should be a main figure.\nResponse: We apologize for the labeling error and thank the reviewer for bringing it to\nour attention. We have corrected the panel labeling in the figure to ensure all elements\nare properly visible. Following the reviewer's valuable suggestion, we have expanded\nthis content and promoted it to a main figure (Revised Fig. 7b) to better accommodate\nand highlight the various conformational states identified in our study. This\nreorganization significantly improves the presentation and impact of our structural\nfindings.\nChange SLC6A to SLC6 though the manuscript\nResponse: We thank the reviewer for this important correction. We have systematically\nreplaced \"SLC6A\" with \"SLC6\" throughout the entire manuscript to maintain\nconsistent and accurate nomenclature for the solute carrier 6 transporter family.\nLine 30: development of “substrate”? substrate analogue perhaps? Or selective\ninhibitors.\nResponse: We thank the reviewer for this precise terminology suggestion. We have\nrevised the text in line 30 to read \"development of taurine transporter substrate\nanalogues or selective inhibitors\" as this more accurately reflects the focus of\ntherapeutic development efforts targeting hTauT.\nLine 35: Remove ‘The’ such that it reads ‘neuromodulation’. Taurine deficiency causes\nfunctional…’\nResponse: We thank the reviewer for this correction. We have removed the word \"The\"\nfrom the beginning of the sentence, which now reads: \"...neuromodulation. Taurine\ndeficiency causes functional...\" as suggested. in line 35.\nLine 38: A reference to knock out mice studies should be added\nResponse: We thank the reviewer for this suggestion. We have added a citation to\nrelevant knockout mouse studies at line 38 in the revised manuscript, supporting the\nstatement about the physiological consequences of taurine deficiency.\nLine 48: Change shares to share\nResponse: According to the reviewer's suggestions, we have corrected the expression\nto “TauT shares >36% sequence similarity with other SLC6 family members, and has\nthe highest sequence identity with hGAT3 (~61%)” in lines 48-50 in the revised\nmanuscript.\nLines 49 – 51 don’t read well, e.g., belong to GABA transporter subfamily?\nResponse: We thank the reviewer for pointing out the lack of clarity in this sentence.\nWe have rewritten the text to clearly state: \"hTauT, along with GAT1 (SLC6A1),\nGAT2\n(SLC6A13), GAT3 (SLC6A11), BGT1 (SLC6A12), and CT1 (SLC6A8), belong to the\nGABA transporter subfamily\" in lines 50-52.\nLine 49: How much similarity is shared? Which member has the highest sequence\nidentity?\nResponse: We thank the reviewer for prompting this clarification. We have now\nspecified in the text that hTauT shares approximately 36% sequence similarity with\nother SLC6 family members and notably has the highest sequence identity (~61%) with\nhGAT3, according to the reviewer's suggestions in lines 48-50.\nLine 51: A comment on the stoichiometry and mentioning of chloride would be\nbeneficial.\nResponse: We thank the reviewer for this suggestion to provide important functional\ncontext. We have revised the text in lines 52-54 to state: \"SLC6s can utilize the\nchemiosmotic gradient of sodium (Na⁺) and chloride (Cl⁻) ions to transport their\nsubstrates across the membrane. For TauT, substrate uptake occurs with a stoichiometry\nof 2 Na⁺:1 Cl⁻.\"\nLine 52: Many members have overlapping substrates, and not all have unique substrates.\nResponse: We thank the reviewer for this important clarification. According to the\nreviewer's suggestions, we have revised the text in lines 52-53 to state: \"SLC6s can\nutilize the chemiosmotic gradient of sodium (Na⁺) and chloride (Cl⁻) ions to transport\ntheir substrates across the membrane”.\nLine 56: Change ‘substrate-structural-similar inhibitors’ to ‘inhibitors that are\nstructurally similar to substrate’\nResponse: We have corrected the expression according to the reviewer's suggestions in\nline 58.\nLine 62: Change ‘substrate binds and transports by TauT’ to ‘substrate binds to and is\ntransported by TauT’\nResponse: We have corrected the expression according to the reviewer's suggestions in\nline 64.\nLine 88-91: Be specific about the inward conformation. Not clear from the text if it is\ninward-open or inward occluded.\nResponse: We have specified the occluded conformation as inward-occluded\nconformation, and the inward conformation as the inward-open conformation according\nto the reviewer's suggestions in the revised manuscript.\nLines 103 – 104 don’t read well.\nResponse: We thank the reviewer for pointing out the lack of clarity in these lines.\nWe\nhave rewritten the passage to improve its flow and precision. The revised text now reads\n(Lines 116-122):\n\"For the apo and P4S-bound states, conformational flexibility of TM1a—initially\nevidenced by discontinuous density—was resolved through 3D variability analysis and\nfocused 3D classification as described above, yielding four well-defined structures.\nAmong these, TM1a is ordered from P39 in the P4S-bound inward-occluded,\nE42 in\nthe P4S-bound inward-open, and apo inward-open conformations. An exception is the\napo inward-occluded structure, in which the N-terminal density for TM1a begins at\nresidue F50 (Extended Data Fig. 6 and Extended Data Table 1).\"\nLines 105 – 116: It is not clear what structure is being discussed here, if it is the general\narchitecture, please clarify that.\nResponse: We thank the reviewer for this important feedback regarding the clarity of\nour structural description. We have revised the relevant section (revised lines 108-114)\nto explicitly state that we are describing the general architecture of hTauT, using the\napo state as a representative example for clarity. To further support this general\narchitectural analysis, we have now included comprehensive local density maps for all\nstructural models in Revised Extended Data Fig. 6, demonstrating the consistent quality\nand features across all determined structures.\nLine 142: It is not clear which residues are TM3 and which are TM6 in the use of\n‘respectively’.\nResponse: We thank the reviewer for highlighting this lack of clarity. We have revised\nthe sentence to explicitly state: \"L134 and Y138 on TM3, and F300, A303, and L306\non TM6\" to clearly associate each residue with its corresponding TM. This revision\nappears in lines 173-175 of the revised manuscript.\nLine 153: Remove ‘smallest’\nResponse: We have removed \"smallest\" according to the reviewer's suggestions in line\n209 in the revised manuscript.\nLines 188 – 190: It is not clear what is the purpose of this sentence, wasn’t this expected\nfrom the chemical structure of P4S?\nResponse: We thank the reviewer for this feedback. The sentence in question was\nindeed redundant, as the chemical structure of P4S inherently suggests its properties as\na substrate analog. We have therefore removed it from the manuscript to improve the\nconciseness and focus of the text.\nLine 191: Rephrase to outline which are main chain interactions and which are side\nchain interactions\nResponse: We thank the reviewer for this suggestion to improve the clarity of our\nstructural descriptions. We have systematically revised the relevant sections throughout\nthe manuscript to explicitly distinguish between main chain and side chain interactions\nin our descriptions of molecular contacts. These clarifications have been incorporated\nin lines 168-171, 175-177, 228-235 and 340-343 of the revised manuscript.\nLine 255: Change ‘away taurine/…’ to ‘away from taurine/…’\nResponse: We thank the reviewer for catching this typographical error. We have\ncorrected the text to read \"away from taurine/β-alanine and P4S\" in line 385 of the\nrevised manuscript.\nLine 256: Rephrase. ‘TM1a moves away from P4S’. It is not clear what the authors are\ntrying to communicate with ‘as taurine/β-alanine’\nResponse: We thank the reviewer for highlighting this lack of clarity. We have rephrased\nthe sentence to read: \"TM1a moves away from both P4S and the physiological\nsubstrates taurine/β-alanine.\" in line 386 of the revised manuscript. This revision\nclarifies that the conformational change of TM1a occurs similarly for these bound\nligands.\nLine 282: BacMam is a virus expression system. ‘transfected’ should be replaced with\n‘transduced’ or further explanation should be used if transfection was used for FSEC\nscreening.\nResponse: We thank the reviewer for this precise terminology suggestion. We have\nrevised the text to clarify our methodology in lines 444-448:\n\"The BacMam plasmids containing the gene of hTauT were small-scale transient\ntransfected into HEK293F cells. The cells were collected after 48 h transfection and\nsolubilized in different detergents to screen detergents and stability of hTauT via\nfluorescence-detection size-exclusion chromatography (FSEC) on the Superose 6\nincrease 5/150 GL column (Cytiva).\"\nThis revision acknowledges the reviewer's correct point while accurately describing our\ntwo-stage process of initial plasmid transfection for screening followed by viral\ntransduction for large-scale expression.\nWe thank the reviewer for the helpful comments. After the gene of hTauT is cloned into\nthe BacMam expression vector, HEK293 cells are small-scale transient transfected with\nthe BacMam plasmid containing the gene of hTauT, and collected after 48h. The cells\nafter transfection are then solubilized in different detergent buffer to screen detergents\nand stability of hTauT by FSEC before moving to the time-consuming process of virus\namplification. We have modified the expression according to the reviewer's suggestions\nin lines 441-451.\n290: replace ‘solubilizing’ with ‘solubilized’\nResponse: We have corrected the expression to “solubilized” according to the\nreviewer's suggestions in line 460.\nLine 292: replace rpm value with g value\nResponse: We have replaced rpm value with g value according to the reviewer's\nsuggestions in lines 456 and 463.\nLine 345: replace ‘cryo-grids’ with ‘cryo-EM grids’\nResponse: We have modified the expression to “cryo-EM grids” according to the\nreviewer's suggestions in lines 564-565.\nOn the methods section:\n278 – 282: It is a very long sentence starting with DNA and ending with protein\nexpression, please break it into shorter sentences for clarity. Similarly, the next section:\nFreeStyle 293F cells at a density of 2.5-3.0 × 106 ml−1 were infected for 48 h with P2\nvirus produced with the Bac-to-Bac system using Sf9 insect cells, and 10 mM sodium\nbutyrate was added to the culture 8-12 h post-infection.\nCan be modified as:\nThe HEK293F cells were typically infected with a xx% volume of infection of the P2\nvirus (please add the titre) at a density of 2.5-3.0 × 106 ml-1. 10 mM sodium butyrate\nwas added to the culture 8-12 h post-infection, and the cells were collected 48 h post-\ninfection by centrifugation at ……\nProduction of virus with Back-to-Bac system can be added as a line in the beginning.\nResponse: We thank the reviewer for these excellent suggestions to improve the clarity\nand completeness of our Methods section. We have thoroughly revised the section as\nfollows (Lines 454-457):\nWe have broken down the long, complex sentences into shorter, more focused\nstatements for better readability. We now clearly state that the Bac-to-Bac baculovirus\nsystem was used to generate the P2 virus in Sf9 insect cells.\nFor the large-scale expression, we have provided the missing details as suggested: \"\nHEK293F cells were typically infected with a 7% (v/v) volume of infection of the P2\nvirus at a density of 2.5-3.0 × 106 ml−1. 10 mM sodium butyrate was added to the culture\n8-12 h post-infection. Cells were collected 48 h post-infection by centrifugation at\n3,500 g for 10 min and washed once with 20 mM Tris-HCl pH 8.0 and 150 mM NaCl.\"\nThese revisions enhance the methodological clarity and reproducibility of our\nexperimental procedures.\nPlease provide as much details as possible: Was the purification performed right after\nharvest? Or were the cells stored in -80 °C.?\nResponse: We thank the reviewer for requesting this important methodological detail.\nWe have updated the manuscript to specify: \" The harvested cells were then flash-frozen\nand stored at −80 °C.\" This clarification has been incorporated into lines 457-458 of the\nrevised Methods section.\nLine 299: Superdex 6 increase 10/300 – superose 6?\nResponse: We thank the reviewer for catching this error. We have corrected the text to\n\"Superose 6 Increase 10/300 GL\" in line 470 in the revised manuscript according to the\nreviewer's kind remind.\nLine 306: How much P2 virus? mention the titre please.\nResponse: We thank the reviewer for highlighting this requirement. We have added the\ntiter in lines 454-455 and line 485-486 as suggested by the reviewer in the revised\nmanuscript.\nThe text now reads:\n“HEK293F cells were typically infected with a 7% (v/v) volume of infection of the P2\nvirus at a density of 2.5-3.0 × 106 ml−1.”\n“HEK293T cells were first seeded into a 6-well plate at a density of 2.0 × 106 cells per\nwell and infected with 0.5% (v/v) P2 virus after culturing for 12 h.”\nLine 306: After infecting for 24 h, can be modified to: 24 h-post infection\nResponse: We have modified this expression to “24 h-post infection” in line 486 in the\nrevised manuscript.\nLine 351: “a defocus of -1.6 μm”, has it been only one defocus value for the taurine-\nbound hTauT data collection, and not a range? If so, please elaborate. The values also\ndiffer from the cryo-EM table, please correct accordingly.\nResponse: We thank the reviewer for catching these inaccuracies. We have corrected\nthe defocus value for the taurine-bound hTauT data collection to properly state the range\nof -1.5 to -1.8 μm in line 570 in the revised manuscript. This correction ensures\nconsistency with the values reported in the cryo-EM data table (Extended Data Table\n1). The revised text now accurately reflects the experimental parameters used during\ndata collection.\nLine 342: flash-frozen --> plunge-frozen\nResponse: We have modified this expression to “plunge-frozen” in line 561.\nLine 387: please reference the non-uniform refinement article as recommended by\ncryoSPARC programmers\nResponse: We thank the reviewer for this important reminder. We have now included\nthe appropriate citation for the non-uniform refinement method (Punjani et al., 2017)\nin line 590 of the revised manuscript, as recommended by the cryoSPARC developers.\nLine 393: please provide the respective alphafold model ID(s).\nResponse: We thank the reviewer for this suggestion to improve the reproducibility of\nour work. We have now provided the specific AlphaFold model identifiers in line 627\nof the revised manuscript.\ntaurine transporter TauT in a human kidney cell line. Pharmacological Reports 71, 121-129\n(2019). https://doi.org/https://doi.org/10.1016/j.pharep.2018.10.005\nSLC6A1 transporter (GAT1). Cellular and molecular life sciences : CMLS 81, 269 (2024).\nhttps://doi.org/10.1007/s00018-024-05309-w\nkidney cells stably expressing the human serotonin transporter. J Pharmacol Exp Ther 293, 870-\n878 (2000).\nhigh-resolution structures of human norepinephrine transporter. Cell Res 34, 810-813 (2024).\nhttps://doi.org/10.1038/s41422-024-01024-0\ntransport mechanism. Nature structural & molecular biology 30, 1023-1032 (2023).\nhttps://doi.org/10.1038/s41594-023-01011-w\ntransporter GAT1. Nature Structural & Molecular Biology 30, 1012-1022 (2023).\nhttps://doi.org/10.1038/s41594-023-00983-z\n2412 (2025). https://doi.org/10.1038/s41467-025-57613-z\nallosteric site. Sci Adv 7, eabl3857 (2021). https://doi.org/10.1126/sciadv.abl3857"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have answered all my questions to full satisfaction.", "reviewer_2": "The manuscript is significantly improved after the revision and the authors have answered most of my queries. The study is now quite extensive and adds substantial information about the mechanisms of substrate selectivity and transport in TauT. A few minor errors like typos are seen. line 245 - decimal for 2.8 angstroms. methods: Explain the procedure followed for 3DVA analyses in the methods. Fig. 3a. beta-Alanine Ki curve is not very clean. Needs to be repeated and a cleaner curve maybe plotted. besides these aspects I think the manuscript is improved from its earlier version.", "reviewer_3": "The authors have addressed the majority of the points. There are still a few issues that need correction/clarification. Please revise accordingly. Major Revisions P4S Inward open structure: There is unaccounted density near F58. When aligning P4S inward open and P4S inward occluded, it seems that TM1a from P4S inward occluded could fit here (particularly F58). Does this mean the particle stack is still mixed and not properly separated? In this model, the density of CHS703 does not support modelling of the CHS molecule, please remove it. Apo inward open There is density that matches where P4S was modelled in the inward open structure (it only disappears at 10 rmsd in Coot). This raises questions about the confidence of P4S in the inward open structure. What could this density represent in the substrate-free map? In the inward occluded apo structure, the two water molecules have been modelled into the density (but it is clearer density there). The density for CLR702 does not support modelling of the CLR molecule, please remove it. Water801 is modelled in 9L1B but density here is only visible at the level of the noise. Please remove it from the model. Whilst the monensin experiments provide compelling evidence that P4S is a substrate, it is still an indirect measure of P4S transport. I would therefore temper the language of line 305 “definitive hallmark” In Extended Fig. 2c, it is not clear why particles of the yellow volume were included, please elaborate (close up screenshot is attached). Line 205: remove (ref) Line 242: In light of the new results presented in the manuscript showing P4S is a substrate, specifying that “P4S acts as a competitive inhibitor of taurine transport by hTauT” may be more appropriate", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-to-point response to reviewer comments\nWe sincerely appreciate the editors and reviewers for their time and insightful\ncomments. Your valuable and constructive comments are of great importance in\nimproving our manuscript. As suggested, we have carefully revised our manuscript\nand performed additional experiments. We have now completed the revision and\naddressed all comments raised by reviewers. Please find the point-to-point responses\nbelow.\nReviewer #1 (Remarks to the Author):\nThe authors have answered all my questions to full satisfaction.\nResponse: We thank the reviewer for their positive feedback and are pleased that our\nresponses and revisions have fully addressed their questions and concerns.\nReviewer #2 (Remarks to the Author):\nThe manuscript is significantly improved after the revision and the authors have\nanswered most of my queries. The study is now quite extensive and adds substantial\ninformation about the mechanisms of substrate selectivity and transport in TauT. A few\nminor errors like typos are seen.\nResponse: We thank the reviewer for their positive assessment of our revised\nmanuscript and are pleased that they find the study now provides substantial\nmechanistic insights into TauT. We are grateful for their constructive feedback\nthroughout the review process, which has significantly strengthened the work.\nIn direct response to their final comment, we have conducted a thorough proofreading\nof the entire manuscript to identify and correct the minor typographical errors. We are\nconfident that this final careful edit has further enhanced the clarity and professionalism\nof the text.\nline 245 - decimal for 2.8 angstroms.\nResponse: We thank the reviewer for their meticulous proofreading. The typographical\nerror on line 245 has been corrected from “2.8 angstroms” to “2.8 Å”.\nmethods: Explain the procedure followed for 3DVA analyses in the methods.\nResponse: We thank the reviewer for this suggestion. We agree that a detailed\ndescription of the 3DVA procedure will enhance the reproducibility of our work. We\nhave now added a dedicated paragraph to the Methods section under “Cryo-EM data\nprocessing” to clearly outline the steps and parameters used, as summarized below.\nTo resolve this heterogeneity, we employed 3D variability analysis (3DVA) in\ncryoSPARC. A consensus refinement map, low-pass filtered to 6 Å, served as the initial\nreference. A soft mask was applied to focus the analysis on the dynamic TM1a region.\nThe 3DVA was computed for 3 modes over a maximum of 20 iterations. Following this,\nparticles were visualized and sorted using the 3DVA display tool in simple mode with\na windowing setting of 2, which organized them into 20 overlapping classes based on\ntheir position along the computed mode. This analysis directly visualized the substantial\nflexibility of TM1a (Extended Data Movies 1-2).\nFig. 3a. beta-Alanine Ki curve is not very clean. Needs to be repeated and a cleaner\ncurve maybe plotted.\nResponse: We thank the reviewer for their careful evaluation of the binding data. We\nacknowledge that the original beta-alanine inhibition curve in Fig. 3a exhibited a degree\nof scatter, which reflects the inherent variability of the transport-based functional assay.\nIn direct response, we repeated the beta-alanine competition experiment in three\nadditional independent replicates. The newly collected data yield a cleaner sigmoidal\ncurve and improve confidence in the fitted parameters (Fig. R1). A slight deviation of\none data point (at 5 µM) from the fit is within the expected experimental variability and\ndoes not impact the overall trend or the derived Ki value.\nWe have now combined all six replicates (original and new) to generate a consolidated,\nmore robust dataset, which has replaced the original panel in Fig. 3a. This revision\nprovides a clearer quantitative representation of the inhibition, confirming beta-\nalanine's role as a competitive inhibitor of hTauT. We are grateful to the reviewer for\nprompting this important validation, which has strengthened our analysis.\nFig. R1 | Inhibition of hTauT by beta-alanine (new data).\nDose–response curve showing beta-alanine inhibition of taurine uptake. Data are\npresented as mean ± SEM (n = 3). The curve was fitted to determine an IC of 64.41\n50\nμM.\nbesides these aspects I think the manuscript is improved from its earlier version.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed the majority of the points. There are still a few issues that\nneed correction/clarification. Please revise accordingly.\nResponse: We thank the reviewer for their continued engagement with our work and\nfor acknowledging that we have addressed the majority of their points. We are\ncommitted to fully incorporating their remaining feedback to ensure the manuscript\nmeets the highest standards.\nMajor Revisions\nP4S Inward open structure:\nThere is unaccounted density near F58. When aligning P4S inward open and P4S\ninward occluded, it seems that TM1a from P4S inward occluded could fit here\n(particularly F58). Does this mean the particle stack is still mixed and not properly\nseparated?\nResponse: We thank the reviewer for this insightful observation regarding the\nunaccounted density near F58 in the P4S-bound inward-open structure, and for raising\nthe critical question of particle stack homogeneity.\nThe reviewer's hypothesis is astute. Our initial reconstruction indeed included particles\nfrom both the well-defined cyan class and the lower-resolution yellow class (Fig. R2a).\nThe yellow class exhibited fragmented TM1a density while retaining some visual\nresemblance to an inward-open conformation. This inclusion likely introduced\nconformational heterogeneity, manifesting as the ambiguous density near F58.\nTo address this, we conducted a dedicated re-analysis. Separate 3D reconstructions\nrevealed a clear distinction: the cyan class displays a continuous TM1a helix, while the\nyellow class shows only fragmented density in this region (Fig. R2a). Volume alignment\nindicates that the two classes are largely similar except for TM1a. In the yellow class,\nTM1a is bent toward the intracellular side and shows a measurable displacement\nrelative to its position in the cyan class (Fig. R2b). Notably, this displaced TM1a density\nmore closely resembles that of the inward-occluded conformation (Fig. R2a). Thus, the\nyellow class appears to represent an inward-occluded-like, rather than a true inward-\nopen conformation.\nConsequently, for the final refinement, we used only particles from the well-defined\ncyan volume, followed by additional 3D classification to remove suboptimal particles.\nThis stringent processing yielded a homogeneous particle set and a final map at ~3.25\nÅ resolution. Crucially, in this revised map, the density near F58 is well-resolved and\nclear (Fig. R2c). The minimal, discontinuous residual density that cannot accommodate\nthe TM1a helix from the inward-occluded conformation, confirming that the initial\nprominent unaccounted density was an artifact of the previously mixed particle set.\nThus, the final structure represents a well-separated, homogeneous conformation. We\nhave updated Extended Data Fig. 5 and its legend.\nThe updated cryo-EM map and model for the P4S-bound inward-open conformation\nhave been deposited in EMDB and PDB under the same accession codes.\nFig. R2 | Revised cryo-EM analysis of the P4S-bound hTauT inward-open\nconformation.\na. Focused 3D classification workflow. The cryo-EM density for TM1a is continuous\nin the cyan class but fragmented in the yellow class. The black box indicates volume\nalignment between the yellow class and the P4S-bound inward-occluded conformation\n(light blue; map contoured at 12.2 σ). The position of TM1a in the yellow volume (black\ncircle) resembles that of the inward-occluded state.\nb. Overall map alignment of the cyan and yellow classes. The arrow indicates the\ndisplacement of TM1a density between the two classes (maps contoured at 12.2 σ).\nc. Comparison of the revised (blue mesh, left) and previous (yellow mesh, right) cryo-\nEM maps of the P4S-bound inward-open conformation, displayed in Coot at two\ncontour levels. Structural models of the P4S-bound inward-open (green) and inward-\noccluded (split-pea) conformations are fitted to the density near F58. Red arrows\nindicate that the improved density in the revised map (contoured at 7.0 σ) cannot\naccommodate TM1a from the inward-occluded conformation.\nIn this model, the density of CHS703 does not support modelling of the CHS molecule,\nplease remove it.\nResponse: We thank the reviewer for their careful scrutiny of the structural model. We\nhave re-examined the revised electron density map for CHS703 and agree that the\ndensity is insufficient to confidently model this molecule. Accordingly, we have\nremoved CHS703 from the model to ensure it accurately reflects the experimental data.\nApo inward open\nThere is density that matches where P4S was modelled in the inward open structure (it\nonly disappears at 10 rmsd in Coot). This raises questions about the confidence of P4S\nin the inward open structure. What could this density represent in the substrate-free map?\nResponse: We thank the reviewer for this exceptionally perceptive question regarding\nthe residual density in the substrate-free, inward-open map. We agree that its\ninterpretation is crucial.\nOur initial reconstruction of the apo inward-open conformation included particles from\nboth the cyan and yellow 3D classes. As detailed in our response concerning Extended\nData Fig. 2c, the yellow class exhibits poorly defined TM1a density and likely\nintroduced conformational heterogeneity, which may account for the ambiguous\ndensity noted by the reviewer.\nFollowing the reviewer’s comment, we performed a detailed re-analysis. The final,\nrefined apo inward-open structure presented in the manuscript was obtained using only\nparticles from the well-defined cyan volume, following additional 3D classification to\nensure homogeneity. This more stringent particle selection yielded a map in which the\ncentral binding site is unambiguous and shows no significant unmodeled density\ncorresponding to the P4S position (even at a contour level as low as 7.8 σ in Coot; Fig.\nR3), despite a modest reduction in resolution (from ~3.18 Å to ~3.33 Å).\nTherefore, we conclude that the residual density in the earlier reconstruction was an\nartifact of the initial, mixed particle set. Its absence in the final, rigorously processed\nmap reinforces both the conformational purity of the apo inward-open state and the high\nconfidence in the modeled P4S ligand within the P4S-bound structure.\nFig. R3 | Ligand-binding site density in the inward-open conformation.\na. Cryo-EM density (gray mesh, contoured at 7.8 σ) for P4S and surrounding residues\n(green) in the P4S-bound inward-open structure (left), compared with the\ncorresponding region in the apo inward-open state (right, empty density), displayed in\nChimeraX.\nb. Equivalent view displayed in Coot, showing the revised density (blue mesh) for the\nP4S-bound state (green model) and the empty density (light pink mesh) for the apo state\n(brown model), both contoured at 7.8 σ.\nIn the inward occluded apo structure, the two water molecules have been modelled into\nthe density (but it is clearer density there).\nResponse: We thank the reviewer for their careful scrutiny of the structural model. As\nnoted in our response regarding Extended Data Fig. 2c, we have reprocessed the data\nfor the apo inward-occluded state. The regenerated electron density map shows no clear\ndensity at the previously modeled water sites. Consequently, water molecules have been\nomitted from these positions in the final model to ensure it accurately reflects the\nexperimental data.\nThe density for CLR702 does not support modelling of the CLR molecule, please\nremove it.\nResponse: We thank the reviewer for their careful scrutiny of the structural model. Upon\nre-examining the revised electron density map, we agree that the density for CLR702\nis insufficient to support its modeling.\nAs detailed in our response regarding Extended Data Fig. 2c, the final reconstruction\nof the apo inward‑occluded state used only particles from the well‑separated cyan\nvolume, which led to a modest reduction in nominal resolution (from ∼3.18 Å to\n∼3.33 Å). In this refined map, the density for CHS703 is also inadequate for reliable\nmodeling due to the lower overall resolution. Consequently, CHS703 has also been\nomitted from the final model.\nThese adjustments ensure that the atomic model accurately reflects the experimental\ndensity.\nWater801 is modelled in 9L1B but density here is only visible at the level of the noise.\nPlease remove it from the model.\nResponse: We thank the reviewer for their meticulous attention to the structural model.\nWe agree with this observation. Upon re-examining the electron density map for the\n9L1B structure, we confirm that the density for Water801 is indeed weak and does not\nmeet the threshold for reliable modeling. In accordance with standard structural\nrefinement practices, Water801 has been omitted from the revised model.\nWhilst the monensin experiments provide compelling evidence that P4S is a substrate,\nit is still an indirect measure of P4S transport. I would therefore temper the language of\nline 305 “definitive hallmark”\nResponse: We thank the reviewer for this insightful comment. We agree that the\nmonensin-based assay, while a powerful and established functional tool, provides\nindirect evidence of transport. Accordingly, we have tempered the language in the\nrevised manuscript (line 375).\nThe phrase \"a definitive hallmark\" has been replaced with \"a key functional\ncharacteristic consistent with SLC6 substrate translocation\".\nThis revision more accurately reflects the inferential nature of the evidence while still\nunderscoring the significance of the observed Na+-dependent, substrate-stimulated\nionophore activity. This change strengthens our manuscript by presenting a more\nprecise interpretation that is fully aligned with the experimental data. We believe this\nadjustment addresses the reviewer's valid concern effectively.\nIn Extended Fig. 2c, it is not clear why particles of the yellow volume were included,\nplease elaborate (close up screenshot is attached).\nResponse: We thank the reviewer for raising this point regarding Extended Data Fig.\n2c, which allows us to clarify our particle selection strategy.\nThe yellow volume, though exhibiting poor density for TM1a, showed some visual\nresemblance to an inward-open conformation (Fig. R4a). To avoid discarding particles\nthat might belong to the inward-open conformation, we initially retained this entire\nclass for further analysis.\nFollowing the reviewer’s comment, we performed a detailed re-analysis. Separate 3D\nreconstructions revealed a clear difference: the cyan class shows a continuous TM1a\nhelix, whereas the yellow class displays only fragmented density in this region (Fig.\nR4a). Volume alignment shows that the two classes are largely similar except for TM1a.\nIn the yellow class, TM1a is bent toward the intracellular side and shows a measurable\ndisplacement relative to its position in the cyan class (Fig. R4b). Notably, this displaced\nTM1a density more closely resembles that of the inward-occluded conformation (Fig.\nR4a).\nThese results demonstrate that the yellow class does not represent a homogeneous\ninward-open state, but rather a heterogeneous ensemble in which TM1a samples a\nposition intermediate toward—or akin to—the inward-occluded conformation.\nConsequently, in the final processing workflow, we used only particles from the well-\ndefined cyan volume for high-resolution refinement, followed by additional 3D\nclassification to remove suboptimal particles.\nWe have updated Extended Data Fig. 2c and its legend. Furthermore, this refined\nprocessing yielded a revised map for the inward-open apo state in which density at the\ncanonical chloride-binding site is absent. Given the lack of supportive density—\npotentially due to the lower resolution or chloride release in this state—we have\nremoved the modeled chloride from the final atomic model. The related figures\n(Revised Figs. 6g and 7g) have been updated with the revised maps and models.\nThe updated cryo-EM map and model for the P4S-bound inward-open conformation\nhave been deposited in EMDB and PDB under the same accession codes.\nFig. R4 | Analysis of conformational heterogeneity in apo inward-open hTauT.\na. Focused 3D classification workflow. The cryo-EM density for TM1a is continuous\nin the cyan class but fragmented in the yellow class. The black box indicates volume\nalignment between the yellow class and the apo inward-occluded conformation (light\norange; maps contoured at 11.9 σ). The position of TM1a in the yellow volume (black\ncircle) resembles that of the inward-occluded state.\nb. Overall map alignment of the cyan and yellow classes (contoured at 11.9 σ). The\narrow highlights the displacement of TM1a density between the two classes.\nLine 205: remove (ref)\nResponse: We thank the reviewer for catching this oversight. The placeholder \"(ref)\"\non line 205 has been removed as requested.\nLine 242: In light of the new results presented in the manuscript showing P4S is a\nsubstrate, specifying that “P4S acts as a competitive inhibitor of taurine transport by\nhTauT” may be more appropriate\nResponse: We thank the reviewer for this insightful suggestion.\nWe have revised the text on line 242 as recommended. This change more precisely\ndescribes P4S's mechanism of action and better integrates the structural findings with\ntheir functional context. We are grateful to the reviewer for this valuable clarification.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for their co-review of this manuscript and for their\nvaluable contribution to the peer-review process."}, {"round": 3, "reviewer_comments": {"reviewer_3": "All of my previous concerns have been adequately addressed in the revised manuscript. I have no further comments."}, "author_response": ""}]

Decision letter