Structural basis of bile acid lateral diffusion facilitated by the OSTα/β transporter

Structural basis of bile acid lateral diffusion facilitated by the OSTα/β transporter | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structural basis of bile acid lateral diffusion facilitated by the OSTα/β transporter Wen-Tao Hou, Kang Xu, Bo-Rui Li, Gaoxingyu Huang, Fang-Fang Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8254570/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract OSTα/β is the essential basolateral transporter responsible for returning reabsorbed bile acids to the portal circulation, yet its molecular mechanism remains unclear. Here, we report the cryo-electron microscopy structure of human OSTα/β at 2.7 Å resolution, revealing a GPCR-like 7-TM architecture distinct from all established SLC transporter folds. We further determined the taurocholate (TC)-bound structure at 3.2 Å, showing that the substrate laterally binds to a surface groove on OSTα, but not in the central cavity. The TC molecule is captured by a line of conserved polar residues along the groove, which were further proved by structure-guided mutagenesis combined with transport assays. Molecular dynamics simulations also suggest that it is energetically favored for TC moving along the surface groove. Together, structural analysis, functional assays, and simulations demonstrate that OSTα/β mediates bile acid transmembrane translocation via a facilitated lateral diffusion mechanism with subtle conformational change, which is distinct from known SLCs. These findings establish a mechanistic framework for OSTα/β function in enterohepatic bile acid circulation and provide a foundation for therapeutic modulation of bile acid homeostasis. Biological sciences/Biochemistry/Proteins/Membrane proteins Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The Organic Solute Transporter α/β (OSTα/β, or SLC51), is a heterodimeric member of the solute carrier (SLC) superfamily, composed of OSTα (SLC51A) and OSTβ (SLC51B), whose transport activity strictly depends on the co-expression of both subunits 1-3 . OSTα/β is highly enriched on the basolateral membranes of intestinal epithelial cells, where it mediates the efflux of bile acids and steroids 1,4 . Beyond bile acids, OSTα/β exhibits a broad substrate spectrum, transporting conjugated steroids and xenobiotics, including clinically relevant compounds such as digoxin, methotrexate and bromosulfophthalein 5-8 . Unlike many other bile acid transporters, such as ABCB11, NTCP, and ASBT 9-11 , OSTα/β mediates sodium- and ATP-independent facilitated diffusion, allowing bidirectional flux driven solely by the electrochemical gradient of its substrates 1,8 . Bile acid transport within the enterohepatic circulation is mediated by a coordinated network of hepatocellular and intestinal transportrs 12 . In hepatocytes, bile acids are taken up from portal blood primarily by the sodium taurocholate co-transporting polypeptide (NTCP) 10,13 and members of the organic anion transporting polypeptide (OATP) family 14,15 , and are subsequently secreted into bile via the canalicular transporter ABCB11, also known as the bile salt export pump (BSEP) 9,16,17 . After facilitating lipid digestion in the intestine, bile acids are reabsorbed across the apical membrane of ileal enterocytes by the apical sodium-dependent bile acid transport (ASBT) 11,13 . Their return from enterocytes to the portal circulation relies almost exclusively on the basolateral transporter OSTα/β, which thus constitutes the indispensable efflux pathway that completes the enterohepatic cycle. This unique localization of OSTα/β at the critical interface between intestinal reabsorption and hepatic uptake underscores its essential role in maintaining systemic bile acid, cholesterol, and lipid homeostasis 12,18 . Therefore, dysfunction of OSTα/β in patient are reported to diagnosed with chronic diarrhea, severe fat-soluble vitamin deficiency or cholestasis 19,20 . Despite its well-established physiological and clinical significance, the molecular mechanism of OSTα/β remains largely elusive 21-25 . The absence of high-resolution structural information has limited fundamental insights into how the two subunits assemble, how substrates are recognized, and how translocation proceeds within the membrane. In particular, how OSTα/β enables passive, and potentially bidirectional transport remains obscure. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of OSTα/β in the apo state and in complex with sodium taurocholate (TC) at 2.7 Å and 3.2 Å resolution, respectively. The structures revealed that OSTα/β forms a dimer-of-heterodimer assemble and harbors an unexpected lateral binding site of bile acids. Together with the transport assays and molecular simulations, our results support a “facilitated lateral diffusion” transmembrane mechanism. These findings provide the first high-resolution structural framework for OSTα/β and uncover an unexpected mode of substrate translocation, thereby redefining the mechanistic basis of organic solute transport in the SLC superfamily. Main Biochemical characterization of OSTα/β We co-overexpressed human OSTα and OSTβ in HEK293F cells, introducing a C-terminal Flag-tag to OSTα, and successfully purified the heterodimeric OSTα/β complex for biochemical characterization. OSTα/β was extracted from membranes using lauryl maltose neopentyl glycol (LMNG) supplemented with cholesteryl hemisuccinate (CHS), followed by detergent exchange into LMNG alone during purification. For surface plasmon resonance (SPR) analysis, we reconstituted the protein into lipid nanodiscs, and measured the binding affinities of purified wild-type OSTα/β to the major bile acids present in the human intestine (Fig 1a). These bile acids share the same steroid nucleus (cholan-24-oic acid scaffold, Fig. 1b). In mammals, the principal primary bile acids are cholic acid (CA) and chenodeoxycholic acid (CDCA), which can be further conjugated with glycine or taurine to generate glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA) 26 . Secondary bile acids, formed by gut microbial transformation, include deoxycholic acid (DCA) and lithocholic acid (LCA), which likewise give rise to conjugated species such as glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), and taurolithocholic acid (TLCA) 27 . OSTα/β exhibited the highest affinities for LCA (60 μM) and DCA (690 μM), followed by CDCA (1.36 mM), GC (2.03 mM), TC (2.16 mM) and CA (3.3 mM). Notably, these affinities inversely correlated with their luminal abundances 28 , suggesting that OSTα/β is intrinsically tuned to accommodate bile acids across a broad concentration range in intestinal epithelial cells. We next performed cell-based transport assays using radiolabeled [³H]-TC. Robust transport activity was observed only for the complete OSTα/β heterodimer, whereas OSTα alone showed no detectable transport (Fig. 1c), consistent with previous reports that the small bitopic subunit OSTβ is indispensable for the functional integrity and membrane stability of OSTα 1-3 . We also detected significant reverse transport of TC, by measuring the amount of retained [³H]-TC after buffer wash relative to the initial loading in the absence or presence of OSTα/β at 0 and 5 min (Fig. 1d). These results demonstrated that OSTα/β functions as a bidirectional facilitated-diffusion transporter that moves substrates down their concentration gradient without requiring external energy input. OSTα/β forms a tight dimer of heterodimers with a unique fold distinct from other SLCs Our biochemical assays indicated that the protein samples are in a physiologically relevant state, which were then applied for cryo-EM data acquisition. We finally solved the structure of OSTα/β at an overall resolution of 2.7 Å using 954,511 particles from the best class (Supplementary Fig. 1a & 2). In total, 9-328 residues of OSTα and 18-64 residues of OSTβ were built into the EM density, except for some unstructured segments. The overall structure of OSTα/β displays a dimer of two heterodimers, which is consist with its size-exclusion chromatography behavior (Supplementary Fig. 1b). Each protomer consists of one molecule of OSTα and one molecule of OSTβ, with OSTα in the center forming the symmetric interfaces (Fig. 2a). OSTα adopts a seven-transmembrane (7-TM) helical architecture, with the helices arranged in a counterclockwise orientation when viewed from the extracellular side (Supplementary Fig. 1d and e). These helices display characteristic tilts and bends that generate tight inter-helical packing reminiscent of class-A GPCRs 29 . The two OSTα molecules form a central lipid-filled cavity at the dimer interface within the membrane (Fig. 2b). This cavity accommodates multiple well-defined densities that could be modeled as cholesterol and phospholipids, which we tentatively fitted as phosphatidic acid (PA) based on the shape of the density. This central cavity is sealed on both the extracellular and intercellular sides. On the extracellular side, side chains from TM4, including Leu114, Leu196, Leu199 and Pro203 neighboring residues, completely occlude the opening (Fig. 2c). On the intracellular side, Pro82, Ile83, and Tyr326 form the constriction that close the other end of the cavity (Fig. 2d). We speculate that lipids within the cavity likely contribute to the tight dimerization by providing additional hydrophobic stabilization. The intracellular loop between TM3 and TM4 (Loop TM3-4 penetrates partially into the membrane, and Cys160-164 and Cys166-167 are palmitoylated, further stabilizing membrane interactions. The N-terminal region of OSTα folds into three short α-helices on the extracellular side, that directly engage the extracellular helix of OSTβ (Fig. 2e). OSTβ itself contains a single transmembrane helix that packs against TM1 and TM7 of OSTα forming a large interface of 1410 Å 2 between two proteins. This interface is strengthened by extensive hydrophobic contacts as well as three specific hydrogen bonds: Arg29 of OSTβ with Gly280 on TM7 of OSTα, Glu23 on the extracellular helix of OSTβ with Arg13 of the OSTα N-terminal loop, and Glu31 of OSTβ with Cys284 of OSTα TM7 (Fig. 2e). The structure of OSTα/β reveals architectural features that diverge substantially from canonical members of SLC superfamily (Supplementary Fig. 3). Most SLC transporters consist of a single polypeptide chain with a self-contained multi-helical transmembrane bundle, typically organized into symmetry-related repeats that enable conformational cycling for alternating access. In contrast, OSTα/β functions as a dimer of heterodimer, in which OSTα contributes a 7-TM bundle while OSTβ provides only a single transmembrane helix, forming a 1:1 complex protomer. Moreover, OSTα lacks the inverted repeat topology and rocker-switch or elevator-like domains characteristic of SLC families such as SLC6, SLC7, SLC17, and SLC25 30-33 . Instead, its 7-TM bundle resembles a GPCR-like helical architecture 29 rather than an SLC transporter fold 34-36 . Together, these features indicate that OSTα/β represents a topologically and mechanistically distinct transporter, employing a heterodimeric 7TM+1TM assembly and a lipid-stabilized dimeric interface unlike any previously characterized SLC topology. The TC-bound structure revealed a facilitated lateral diffusion mechanism for substrate translocation To determine the transport mechanism of this unique member of SLCs, we solved the TC-bound OSTα/β structure by incubating the protein with 700 μM TC, at a resolution of 3.2 Å (Fig. 3a, Supplementary Fig. 4). Although processed without imposed symmetry (C1), two TC densities were observed symmetrically on opposite sides of OSTα at positions previously occupied by cholesterol molecule in the apo structure (Fig. 2b). The TC molecule occupies the surface-exposed groove between TM5 and TM6 of OSTα. Surface electrostatic analysis revealed a positively charged niche at the intracellular entrance of this groove, where Arg241 and Arg244 interact with the sulfonate moiety of TC via electrostatic interactions, including a salt bridge (Fig. 3b). In addition, Gln260 forms a hydrogen bond with the carbonyl group of TC. The TC molecule is further stabilized by hydrophobic interaction contributed mainly by the membrane-inserted Loop TM3-4 and its palmitoylated segment, forming a semi-open pocket embedded within the lipid bilayer (Fig. 3b). Superposition of the TC-bound and apo structures yielded an RMSD of 0.663 Å over 699 residues aligned Cα atoms, showing minimal backbone rearrangement and arguing against classical conformational change-based mechanisms used by many SLC transporters, such as rocker-switch, elevator, or alternating-access modes 34-36 . Nevertheless, subtle but meaningful conformational changes are observed in residues that interact with TC, particularly near the entry site: the side chain of Arg241 rotates by 4.9 Å relative to its position in the apo structure, avoiding the steric clash while also keeping an electrostatic interaction with the sulfonate moiety of TC (Fig. 3c). Along the lateral groove, several additional polar residues, such as Lys191, Asn222, Thr229, and Ser271, extend from the binding site toward the membrane and, together with several hydrophobic residues from TM5 and TM6, form an amphipathic diffusion path (Fig. 3d). Sequence alignment demonstrated that these residues, including those directly coordinating TC and those lining the putative diffusion path, are highly conserved among OSTα homologues (Fig. 3e). Consistently, transport assays using radiolabeled [³H]-TC combined with site-directed mutagenesis revealed that mutations in the binding site (R241A and R244A) as well as residues along the proposed diffusion path (e.g., K191A) markedly reduced transport activity (Fig. 3f). In addition, deletion of the palmitoylated segment of Loop TM3-4 showed a significant reduced transport activity, indicating its important role in consisting the semi-open pocket embedded within the lipid bilayer. To further evaluate the existence of this diffusion path, we performed molecular dynamics (MD) simulations to estimate the energy required for a TC molecule to traverse either the central cavity or the lateral surface groove. The calculations revealed that TC passage through the lateral surface groove displays a much lower energy barrier than passage through the central cavity, which is highly energetically unfavorable (Fig. 4a). Moreover, our transport activity assays with site-directed mutagenesis also indicated that the central cavity does not function as a transport path (Supplementary Fig. 5). Together, our results strongly support a facilitated lateral diffusion mechanism, in which bile acids diffuse across the membrane along the surface groove of OSTα/β, driven solely by their concentration gradient. Discussion OSTα/β is the principal basolateral bile acid exporter in ileal enterocytes and a key determinant of enterohepatic bile acid circulation, yet its transport mechanism has remained unresolved despite decades of physiological, biochemical and clinical work. Here we provide high-resolution structures of human OSTα/β together with comprehensive functional analysis, and show that this heterodimeric SLC operates through an unexpected facilitated lateral diffusion mechanism. Rather than forming a classical translocation pore, OSTα/β uses a membrane-embedded, surface-exposed groove to mediate sodium- and ATP-independent, bidirectional diffusion of bile acids along their concentration gradient. Alternating-access, rocker-switch or elevator-type transporters typically rely on substantial rearrangements of transmembrane helices to alternately expose a central binding site to opposite sides of the membrane 34-36 . By contrast, superposition of our apo and TC-bound structures reveals minimal backbone rearrangement, with ligand binding accommodated largely by local side-chain adjustments around the entry site. The two OSTα subunits enclose a lipid-filled central cavity that is sealed on both extracellular and intracellular sides, structurally incompatible with a water-filled translocation pathway. Instead, TC binds in a lateral surface groove at the OSTα membrane interface, coordinated by conserved basic and polar residues and buttressed by a palmitoylated Loop TM3-4 that shapes a semi-open pocket within the bilayer. Therefore, structural analysis combined with biochemical assays and MD simulation, we can depict the essential snapshots of how substrates are laterally diffused facilitated by OSTα/β (Fig. 4b, Supplementary Movie 1). In the physiological situation, bile acids were absorbed by ASBT in a sodium-dependent manner from the intestinal into enterocyte, then carried by IBABP towards the basolateral membrane where OSTα/β localized (Fig. 4c). As result, there is usually a higher concentration of bile acids than portal blood. Therefore, the facilitated diffusion should normally start from semi-open pocket within the bilayer, as we captured in the TC-bound structure (Snapshot 1). Along the amphiphilic groove towards the portal blood, conserved residues lead the flow of bile acids outwards, especially Lys191, as confirmed by our assays (Snapshot 2). Afterwards, bile acids would continue to move along the groove until it meets Loop TM4-5 . According to the simulation, energies provided by the gradient is sufficient to expel the loop for the move on of bile acids (Snapshot 3). Mutational disruption of key residues in this groove strongly impairs diffusion, and MD simulations demonstrate that diffusion along this lateral pathway is energetically much more favorable than passage through the central cavity. Together, these observations support a model in which bile acids remain largely within the lipid phase and diffused laterally along an amphipathic surface groove, rather than being alternately buried and exposed in a central aqueous cavity. Beyond establishing a mechanistic model for OSTα/β itself, our work has broader implications for the SLC superfamily and for membrane transport paradigms in general. OSTα/β possesses a GPCR-like 7-TM architecture and a dimer-of-heterodimers organization that are distinct from the major SLC folds described to date, and uses a membrane-embedded lateral groove rather than a deeply buried central cavity to translocate amphipathic substrates. OSTβ appears to act as a structural brace that locks the 7-TM core of OSTα into a competent conformation and helps organize the extracellular architecture, without directly contributing to the lateral transport groove. Given the central position of OSTα/β at the interface between intestinal reabsorption and hepatic uptake, the structural framework presented here provides a rational basis for designing small molecules that modulate its activity, with potential applications in cholestatic liver disease, bile acid diarrhoea and metabolic disorders driven by altered bile acid signaling. Materials and Methods Protein expression and purification The full-length human OSTα gene (UniProt ID: Q86UW1) and OSTβ gene (UniProt ID: Q86UW2) were synthesized by Sangon Biotech Company with codon optimization for eukaryotic expression. The OSTα was cloned into a pCAG vector with a C-terminal Flag tag (DYKDDDDK) using the ClonExpress® II One Step Cloning Kit (Vazyme Biotech Co., Ltd). The OSTβ was subcloned into a pCAG vector without any tag. Using homologous recombination of multiple fragments, all point mutations were introduced by designing forward and reverse primers with point mutations and overlapped amplifying fragments. For protein expression, HEK293F cells were cultured in SMM 293T-II medium (Sino Biological Inc.) at 37℃ and 130 rpm with 5% CO 2 . Cells were transfected when the density reached 2.5~3.0 × 10 6 cells per mL. For cell transfection, 1.5 mg OSTα expression plasmid and 1.5 mg OSTβ expression plasmid were premixed with 6 mg linear polyethylenimine (PEI) (Polysciences, Inc) of 25,000 MW in 45 mL fresh medium for 15 min, then the mixture was added to 800 mL HEK293F cells, supplemented with another 45 mL medium, and incubated once more for 15 min at rest. The transfected cells were grown at 37℃ for 48-60 h before harvesting. Cell pellets were resuspended in the lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl (for the SPR experiment, the buffer system was adjusted to 50 mM HEPES-KOH pH 7.5, 150 mM NaCl, the same below) and 20% (v/v) glycerol after centrifugation at 5,000 rpm for 7 min. The suspension was flash-frozen in liquid nitrogen and stored at -80℃ for further use. For protein purification, the collected cells were lysed using an AH-1500 High Pressure Homogenizer (ATS inc.) with 5 passes at ~600 bar, followed by centrifugation at 17,300 × g for 20 min to remove the cell debris. The supernatant was subjected to ultracentrifugation at 45,000 rpm for 45 min (Beckman, Type 70 Ti). The membrane precipitates were collected and incubated with the lysis buffer plus additional 1% (w/v) lauryl maltose neopentyl glycol (LMNG, NG310, Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS, C6013-25, Anatrace) at 6°C for 2 h. Insoluble material was removed by ultracentrifugation at 45,000 rpm for 45 min (Beckman, Type 70 Ti) at 4℃. The supernatant was incubated in a decolorization shaker with the anti-FLAG M2 affinity gel (Sigma) on ice for 1 h. The resin was then loaded onto the column and washed six times, each time with 5 mL of wash buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG. Protein was eluted in batches to 6 mL after 15 min incubation with elution buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG plus 200 μg/mL FLAG peptide. The eluate was then collected and concentrated using a 100-kDa MWCO Amicon Ultra centrifugal filter (Millipore) before being applied to size-exclusion chromatography by a Superose 6 Increase 10/300 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG. Peak fractions were pooled and flash-frozen in liquid nitrogen for future functional experiments or concentrated for nanodiscs reconstitution. All steps were performed either in cold storage or on ice. Nanodisc preparation For nanodisc preparation, the concentrated OSTα/β complex sample was reconstituted into lipid nanodiscs by mixing with purified MSP1D1 scaffold protein and porcine brain polar lipid extract (BPL, Avanti) at a 1:4:240 molar ratio. After 1 h of incubation on ice with shaking, 800 mg/mL Bio-Beads SM-2 resin (Bio-Rad) was added to the mixture to initiate the nanodiscs reconstitution reaction by removing the detergent. The mixture was incubated overnight in cold storage with constant rotation. The bio-beads were removed, and the mixture was cleared of debris by centrifugation. The supernatant was loaded onto a Superose 6 Increase 10/300 GL column in 50 mM HEPES-KOH pH 7.5, 150 mM NaCl buffer without detergent. Peak fractions were collected and concentrated for biochemical studies. Surface plasmon resonance (SPR) experiments All SPR experiments were performed on a Biacore 8000 instrument (Cytiva) at 25°C in buffer of 50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 5% DMSO. The purified OSTα/β in lipid nanodiscs was covalently immobilized onto the series S CM5 sensor chips (Cytiva) by amine-coupling chemistry. Different types and concentrations of bile acid in the running buffer were serially flowed over the chip surface with the immobilized protein and the blank for 1 min at a flowrate of 30 μL min -1 . The data were fitted with steady-state affinity binding model using Biacore Insight Evaluation software. The graphs were plotted using OriginPro 2023 software. Cell-based taurocholic acid transport assays The wild type or mutants of OSTα/β were subcloned into the pCAG vector, respectively. HEK293F cells at a density of 1.5 × 10 6 cells per mL were transfected with the empty vector or OSTα/β constructs. 24 h after transfection, cells were collected by centrifugation and resuspended for the Hank’s Balanced Salt Solution (137 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl 2 , 0.81 mM MgSO 4 , 0.49 mM MgCl 2 , 4.17 mM NaHCO 3 , 0.34 mM Na 2 HPO 4 , 0.44 mM KH 2 PO 4 , 0.44 mM D-Glucose). Cell counts were determined using Coulter counting and microscopic visualization. For all assay system, a 200 μL aliquot of cell suspension contains 3 × 10 6 cells. For the [ 3 H] taurocholic acid accumulation assay, cells were resuspended and incubated at 37°C with the HBSS buffer, containing 50 nM [ 3 H] taurocholic acid (specific activity 20 Ci mmol −1 , American Radiolabeled Chemicals). The loading process was stopped by centrifugation at indicated time points. Cells were then washed twice with the ice-cold HBSS buffer with 5 μM taurocholic acid, and resuspended with the same buffer plus 1% Triton X-100 for cell lysis. The radioactivity in the cell lysis was counted using liquid scintillation counting (Tri-Carb 2910TR, PerkinElmer). For the taurocholic acid efflux assay, cells were first loaded in HBSS buffer, plus 50 nM [ 3 H] taurocholic acid for 10 min, then washed and resuspended with [ 3 H] taurocholic acid-free HBSS buffer. 200 μL aliquots were taken immediately after resuspension (defined as the zero time point) or at other indicated time points. Cells were centrifuged and washed twice with 1 mL ice-cold HBSS buffer, and resuspended with the same buffer plus 1% Triton X-100 for scintillation counting. Cryo-EM sample preparation and data acquisition For the wild-type sample, 3.5 μL of purified OSTα/β at a concentration of ~2 mg/mL was applied to glow-discharged holey carbon grids QUANTIFOIL (Au R2/1, 300-mesh,). The grids were blotted with filter paper for 3.5 s and zero blotting force. Then, the grids were plunged into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (FEI) under 100% humidity at 8°C. A total of 9,788 micrograph stacks were automatically collected with EPU 2 software on a Titan Krios 300 kV transmission electron microscope equipped with a K3 Summit direct electron detector (Gatan) and a GIF Quantum energy filter (Gatan) at a defocus range of -2.3 to -1.3 μm with a magnification of ×105,000, resulting in a pixel size of 0.82 Å. Each movie stack containing 32 frames was exposed in a super-resolution mode, with a total dose of 55 e - /Å 2 . To obtain the structure of the sodium taurocholate bound OSTα/β complex, 500 μM TC was maintained in the buffer throughout the protein purification process. The purified protein concentrated to ~3 mg/ml was incubated with 200 μM sodium taurocholate on ice for 30 min. After that, aliquots of 3.5 μL protein complex were applied to glow-discharged holey carbon grids QUANTIFOIL (Au R2/1, 300-mesh,) The grids were blotted with filter paper with a 3.5 s blotting time and zero blotting force. Then the grids were plunged into liquid ethane cooled with liquid nitrogen using a Vitrobot Mark IV (FEI) under 100% humidity at 8°C. A total of 6,137 micrograph stacks were collected in the same manner as described above. Cryo-EM data processing Dose-fractionated image stacks were applied to motion correction and contrast transfer function estimation, which are both implemented in cryoSPARC 4.7.1 37 . Particles were automatically picked with template picker or topaz pick and extracted for further 2D and 3D processing. For OSTα/β, first, 2D classification was performed using particles blob picked from the two datasets. The top 59,926 particles were selected as template to template picking, followed by multiple rounds of 2D classification. The selected 1,375,076 high-quality particles were subjected to ab-initio reconstruction, and the best class was chosen for homogeneous refinement and NU refinement. Subsequently, template picking and Topaz picking were conducted using all micrographs. After multiple rounds of 2D classification, ab-initio reconstruction and heterogeneous, 954,511 particles were further subjected to homogeneous refinement and NU refinement, yielding a reconstruction map at an average resolution of 2.68 Å (Fig. S2). For TC bound OSTα/β, the workflow is similar to the above. A total of 6,137 movies were used for calculation, and finally, 146,494 particles were further subjected to homogeneous refinement and NU refinement, yielding a reconstruction map at an average resolution of 3.20 Å (Fig. S3). Model building and refinement The final local refinement map was used for model building in Coot. Using UCSF Chimera, the initial rigid body model of OSTα/β predicted by Alphafold 2 38 , was docked into the construction map. Then, model building and refinement were performed manually by Coot. After several rounds of manual refinement, the model was improved by iterative cycles of refinement using real_space_refine in Phenix 39,40 . The final model was validated by phenix.validation_cryoem, and all the figures were prepared with PyMOL (https://pymol.org) or ChimeraX 41 . Well-tempered meta-dynamics (WT-MTD) simulations CHARMM-GUI 42-44 website was utilized to embed the protein, solvate the complex and generate the force field. The complexes were embedded into a bilayer consist of 70% palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules with 30% cholesterol and solvate with the TIP3P model and 150 mM KCl at PH 7.0. Proteins of complexes were described by CHARMM36m forcefield, and the other ligands’ parameters were generated by CGenFFin CHARMM-GUI 45,46 . All of the MD simulations were performed with GROMACS 2025.2 at a constant temperature of 310 K. Each of simulations was started with energy-minimized, and then run 750 ps NVT equilibration steps with positional restraints. Next, 100 ns NVT unrestrained production simulation were operated. The equilibration of the systems was confirmed through monitoring time-dependent RMSD of the complexes. The protein-ligand complex, membrane, and solvent were coupled separately to a temperature bath of 303.15 K using the velocity-rescaling thermostat with a relaxation time of 1.0 ps. All covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing stable integration at the chosen time step. Van der Waals interactions were treated using a force-switch cutoff scheme with switching starting at 1.0 nm and a cutoff at 1.2 nm. The neighbor list was updated every 20 steps using the Verlet cutoff scheme. Electrostatic interactions were computed using the particle mesh Ewald (PME) method with a real-space cutoff of 1.2 nm. Steered Molecular Dynamics (SMD) simulations were performed using the PLUMED plugin. The reaction coordinate (Collective Variable, CV) was defined as the z-component of the center of mass position of the TCH molecule. Moving restraint was applied with a velocity of 1 Å·ns -1 and a moving force constant k from 0 to 200 kJ·mol -1 ·nm -2 . WT-MTD production simulations 47 were using the same conditions applied during equilibration. An integration time step of 4 fs was employed. Two collective variables (CVs) were chosen: the z-component of the center of mass of the TCH molecule (excluding hydrogens) and the Euclidean distance between S of TCH and N of Lys191’s side chain. A well-tempered bias was applied with Gaussian widths of 0.10 nm (for both CVs), an initial height of 2.0 kJ·mol⁻¹, and a bias factor of 15. Harmonic upper and lower walls were applied to restrict the sampling space to the physically relevant region. Declarations Acknowledgment We thank Dr. Yong-Xiang Gao at the Center for Integrative Imaging, University of Science and Technology of China during cryo-EM image acquisition. This work was supported by the Ministry of Science and Technology of China (2024YFA1307900 to W.-T.H, 2024YFA1306103 to Y.C) and the Ministry of Science and Technology of China (2024YFA1306103 to Y.C), National Natural Science Foundation of China (32371257 to Y.C) and USTC Research Funds of the Double First-Class Initiative (YD9100002057 to W.-T.H), Author contributions K.X., Y.C. and W.-T.H. conceptualized this study. C.-Z.Z and Y.C. supervised the project. K.X., and W.-T.H. designed all the experiments. K.X., C.-Y.C. and G.-T.C. performed cloning. K.X., and F.-F.W. performed biochemical characterization. K.X. performed expression, purification, cryo-EM sample preparation, screening, cryo-EM data collection and model refinement. K.X. and G.H. performed structure determination. K.X. and B.-R.L. performed MD simulations. K.X., J.W. and B.W. performed data analysis. K.X., W.-T.H., C.-Z.Z., and Y.C. wrote the manuscript. Data Availability The cryo-EM density maps of two structures have been deposited at the Electron Microscopy Data Bank under accession codes: EMD-XXXX for OSTα/β, EMD-XXXX for TC-bound OSTα/β, and the coordinates have been deposited at the Protein Data Bank under accession codes: XXXX for OSTα/β and XXXX for TC-bound OSTα/β. Declaration of interests The authors declare no competing interests. References Dawson, P.A. et al. The Heteromeric Organic Solute Transporter α-β, Ostα-Ostβ, Is an Ileal Basolateral Bile Acid Transporter*. Journal of Biological Chemistry 280 , 6960-6968 (2005). Li, N., Cui, Z., Fang, F., Lee, J.Y. & Ballatori, N. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8254570","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":573387035,"identity":"6534c595-d585-4c91-a56f-ec1d52de5e67","order_by":0,"name":"Wen-Tao 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CDCA: chenodeoxycholic acid; DCA: deoxycholic acid; CA: cholic acid; LCA: lithocholic acid; TC: taurocholic acid; GC: glycocholic acid. \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values were determined by steady-state affinity analysis. RU, response unit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. The general structural formula of bile acids, with the groups at different positions of the aforementioned six bile acids labeled in the figure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Relative accumulation of [³H]-taurocholic acid at 10 min in cells expressing empty vector, OSTα alone, or the OSTα/β complex. The influx activity of the empty vector was set to 100%. One‐way analysis of variance (ANOVA) is used for the comparison of statistical significance of OSTα with the OSTα/β complex. The\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u0026nbsp;values of \u0026lt; 0.001 are indicated with ***, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e [\u003csup\u003e3\u003c/sup\u003eH] taurocholic acid retention after buffer wash relative to the initial loading in the absence or presence of OSTα/β measured at 0 and 5 min to evaluate its efflux activity.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/ac2c14cf6b836218e957da1a.png"},{"id":101488000,"identity":"84bf4abd-a1e2-42db-8ffa-8121a126a573","added_by":"auto","created_at":"2026-01-30 09:34:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1205646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall architecture of OSTα/β and interaction interfaces.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Overview of the electron density of OSTα/β.Densities corresponding to OSTα are colored in blue and OSTβ are in pink, cholesterol (CHOL) and phosphatidic acid (PA) are colored in orange, S-palmitoyl modification on the cysteine side chains are colored in red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Cartoon representation of the OSTα/β complex structure. Dark blue and light blue denote each OSTα monomer, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Zoom-in presentation of the extracellular side of OSTα-OSTα interface. The interaction residues are shown as sticks, and hydrogen bonds and salt bridges are shown as dotted lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Zoom-in presentation of PA and CHOL in the central cavity of the OSTα-OSTα interface.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee. \u003c/strong\u003eZoom-in presentation of one OSTα-OSTβ interface. The interaction residues are shown as sticks, and hydrogen bonds and salt bridges are shown as dotted lines.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/a4068514678644e2dec58f02.png"},{"id":101488003,"identity":"c94568ff-5dc4-412f-90f0-207da755584c","added_by":"auto","created_at":"2026-01-30 09:34:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":942353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and functional analysis of TC-bound OSTα/β\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Cartoon representation of the OSTα/β complex structure. Dark blue and light blue denote each OSTα monomer, taurocholate (TC) molecules are colored by green. The surface of OSTα/β is colored by the electrostatic potential calculated in Pymol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. The binding pocket of the TC. The binding residues are shown as sticks, and hydrogen bonds and salt bridges are shown as dotted lines. “S-pal” indicated S-palmitoyl modification on the cysteine side chain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Superposition of apo-form and TC-bound OSTα/β structures showing TC or cholesterol superimposed at the same binding pocket. In the apo-form and TC bound form. Arg241 undergoes rotation from the apo-form structure (shown as white sticks) to TC-bound structure (shown as blue sticks) for ~4.9 Å. Comparably, Arg244 can be perfectly superimposed from the two structures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e. Electrostatic surface representation of the semi-open TC-binding pocket located within the lateral groove of OSTα in the lipid bilayer\u003cstrong\u003e. \u003c/strong\u003eThe side chains of possible coordinating residues are shown as stick.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e. The multiple-sequence alignment of the binding pocket and the transport groove. Transmembrane helices are shown as columns in blue.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e. Transport activities of [³H] taurocholic acid mediated by the wild-type OSTα/β complex and OSTα/β mutants at putative key substrate-interacting residues. The activity of the wild type was set to 100%. One‐way analysis of variance (ANOVA) is used for the comparison of statistical significance of wild-type OSTα/β (WT) with its mutants. The P values of \u0026lt; 0.05, 0.01, and 0.001 are indicated with *, **, and ***, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/c32d0d20bfb190ecd432d15b.png"},{"id":101487997,"identity":"cb720bd8-3a3a-4e83-8c23-cba6eb1fdb1f","added_by":"auto","created_at":"2026-01-30 09:34:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":493005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFacilitated lateral diffusion mechanism of OSTα/β-mediated substrate translocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. MD simulations to estimate the energy required for a TC molecule to traverse the central cavity or the lateral surface groove. The results show that the energy required for the central cavity is much higher when approaching the outer membrane leaflet than that for the lateral surface groove.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. Three snapshots of TC facilitated diffusion from enterocyte to the portal blood. The lateral groove is shown in electrostatic surface representation, and three snapshots (indicated as ①, ②, ③)are illustrated. Coordinated residues in the groove are shown as sticks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. A diagram of how bile acids transported from intestinal lumen to portal blood. ASBT (PDB ID: 3ZUX) transports bile acids from the intestinal lumen to the side of the intracellular apical membrane in a sodium dependent manner. Subsequently, IBABP (PDB ID: 3ELX) binds to free bile acids and transports them to the basolateral membrane side. Finally, OSTα/β facilitated the diffusion of bile acids to the portal blood, which is dependent solely on the concentration gradient of bile acids. Numbers on OSTα/β corresponds to the three snapshots shown in \u003cstrong\u003eb\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/9e42d977001e905e2a49c203.png"},{"id":101751687,"identity":"104be171-c595-47e5-94e0-d68680e15d4a","added_by":"auto","created_at":"2026-02-03 10:22:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3868047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/6fed5dad-8db7-48dc-8384-7ee103de705a.pdf"},{"id":101488001,"identity":"ab64b271-3e77-4c69-a847-2bb17b10428b","added_by":"auto","created_at":"2026-01-30 09:34:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3004844,"visible":true,"origin":"","legend":"supplymentary","description":"","filename":"supplymentary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/eddcc3c6b0eee8c9e39df7f3.docx"},{"id":101487999,"identity":"7430fd99-f01f-4be9-ab78-3131c00f84b6","added_by":"auto","created_at":"2026-01-30 09:34:26","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2393676,"visible":true,"origin":"","legend":"movie","description":"","filename":"supplementarymovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8254570/v1/459053fb78e2c7e9873d7912.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural basis of bile acid lateral diffusion facilitated by the OSTα/β transporter","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Organic Solute Transporter \u0026alpha;/\u0026beta; (OST\u0026alpha;/\u0026beta;, or SLC51), is a heterodimeric member of the solute carrier (SLC) superfamily, composed of OST\u0026alpha; (SLC51A) and OST\u0026beta; (SLC51B), whose transport activity strictly depends on the co-expression of both subunits\u003csup\u003e1-3\u003c/sup\u003e. OST\u0026alpha;/\u0026beta; is highly enriched on the basolateral membranes of intestinal epithelial cells, where it mediates the efflux of bile acids and steroids\u003csup\u003e1,4\u003c/sup\u003e. Beyond bile acids, OST\u0026alpha;/\u0026beta; exhibits a broad substrate spectrum, transporting conjugated steroids and xenobiotics, including clinically relevant compounds such as digoxin, methotrexate and bromosulfophthalein\u003csup\u003e5-8\u003c/sup\u003e. Unlike many other bile acid transporters, such as ABCB11, NTCP, and ASBT\u003csup\u003e9-11\u003c/sup\u003e, OST\u0026alpha;/\u0026beta; mediates sodium- and ATP-independent facilitated diffusion, allowing bidirectional flux driven solely by the electrochemical gradient of its substrates\u003csup\u003e1,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBile acid transport within the enterohepatic circulation is mediated by a coordinated network of hepatocellular and intestinal transportrs\u003csup\u003e12\u003c/sup\u003e. In hepatocytes, bile acids are taken up from portal blood primarily by the sodium taurocholate co-transporting polypeptide (NTCP)\u003csup\u003e10,13\u003c/sup\u003e and members of the organic anion transporting polypeptide (OATP) family\u003csup\u003e14,15\u003c/sup\u003e, and are subsequently secreted into bile via the canalicular transporter ABCB11, also known as the bile salt export pump (BSEP)\u003csup\u003e9,16,17\u003c/sup\u003e. After facilitating lipid digestion in the intestine, bile acids are reabsorbed across the apical membrane of ileal enterocytes by the apical sodium-dependent bile acid transport (ASBT)\u003csup\u003e11,13\u003c/sup\u003e. Their return from enterocytes to the portal circulation relies almost exclusively on the basolateral transporter OST\u0026alpha;/\u0026beta;, which thus constitutes the indispensable efflux pathway that completes the enterohepatic cycle. This unique localization of OST\u0026alpha;/\u0026beta; at the critical interface between intestinal reabsorption and hepatic uptake underscores its essential role in maintaining systemic bile acid, cholesterol, and lipid homeostasis\u003csup\u003e12,18\u003c/sup\u003e. Therefore, dysfunction of OST\u0026alpha;/\u0026beta; in patient are reported to diagnosed with chronic diarrhea, severe fat-soluble vitamin deficiency or cholestasis\u003csup\u003e19,20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite its well-established physiological and clinical significance, the molecular mechanism of OST\u0026alpha;/\u0026beta; remains largely elusive\u003csup\u003e21-25\u003c/sup\u003e. The absence of high-resolution structural information has limited fundamental insights into how the two subunits assemble, how substrates are recognized, and how translocation proceeds within the membrane. In particular, how OST\u0026alpha;/\u0026beta; enables passive, and potentially bidirectional transport remains obscure.\u003c/p\u003e\n\u003cp\u003eIn this study, we determined the cryo-electron microscopy (cryo-EM) structures of OST\u0026alpha;/\u0026beta; in the apo state and in complex with sodium taurocholate (TC) at 2.7 \u0026Aring; and 3.2 \u0026Aring; resolution, respectively. The structures revealed that OST\u0026alpha;/\u0026beta; forms a dimer-of-heterodimer assemble and harbors an unexpected lateral binding site of bile acids. Together with the transport assays and molecular simulations, our results support a \u0026ldquo;facilitated lateral diffusion\u0026rdquo; transmembrane mechanism. These findings provide the first high-resolution structural framework for OST\u0026alpha;/\u0026beta; and uncover an unexpected mode of substrate translocation, thereby redefining the mechanistic basis of organic solute transport in the SLC superfamily.\u003c/p\u003e"},{"header":"Main","content":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of OST\u0026alpha;/\u0026beta;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe co-overexpressed human OST\u0026alpha; and OST\u0026beta; in HEK293F cells, introducing a C-terminal Flag-tag to OST\u0026alpha;, and successfully purified the heterodimeric OST\u0026alpha;/\u0026beta; complex for biochemical characterization. OST\u0026alpha;/\u0026beta; was extracted from membranes using lauryl maltose neopentyl glycol (LMNG) supplemented with cholesteryl hemisuccinate (CHS), followed by detergent exchange into LMNG alone during purification. For surface plasmon resonance (SPR) analysis, we reconstituted the protein into lipid nanodiscs, and measured the binding affinities of purified wild-type OST\u0026alpha;/\u0026beta; to the major bile acids present in the human intestine (Fig 1a). These bile acids share the same steroid nucleus (cholan-24-oic acid scaffold, Fig. 1b). In mammals, the principal primary bile acids are cholic acid (CA) and chenodeoxycholic acid (CDCA), which can be further conjugated with glycine or taurine to generate glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA)\u003csup\u003e26\u003c/sup\u003e. Secondary bile acids, formed by gut microbial transformation, include deoxycholic acid (DCA) and lithocholic acid (LCA), which likewise give rise to conjugated species such as glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), glycolithocholic acid (GLCA), and taurolithocholic acid (TLCA)\u003csup\u003e27\u003c/sup\u003e. OST\u0026alpha;/\u0026beta; exhibited the highest affinities for LCA (60 \u0026mu;M) and DCA (690 \u0026mu;M), followed by CDCA (1.36 mM), GC (2.03 mM), TC (2.16 mM) and CA (3.3 mM). Notably, these affinities inversely correlated with their luminal abundances\u003csup\u003e28\u003c/sup\u003e, suggesting that OST\u0026alpha;/\u0026beta; is intrinsically tuned to accommodate bile acids across a broad concentration range in intestinal epithelial cells.\u003c/p\u003e\n\u003cp\u003eWe next performed cell-based transport assays using radiolabeled [\u0026sup3;H]-TC. Robust transport activity was observed only for the complete OST\u0026alpha;/\u0026beta; heterodimer, whereas OST\u0026alpha; alone showed no detectable transport (Fig. 1c), consistent with previous reports that the small bitopic subunit OST\u0026beta; is indispensable for the functional integrity and membrane stability of OST\u0026alpha;\u003csup\u003e1-3\u003c/sup\u003e. We also detected significant reverse transport of TC, by measuring the amount of retained [\u0026sup3;H]-TC after buffer wash relative to the initial loading in the absence or presence of OST\u0026alpha;/\u0026beta; at 0 and 5\u0026thinsp;min (Fig. 1d). These results demonstrated that OST\u0026alpha;/\u0026beta; functions as a bidirectional facilitated-diffusion transporter that moves substrates down their concentration gradient without requiring external energy input.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOST\u0026alpha;/\u0026beta; forms a tight dimer of heterodimers with a unique fold distinct from other SLCs \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur biochemical assays indicated that the protein samples are in a physiologically relevant state, which were then applied for cryo-EM data acquisition. We finally solved the structure of\u0026nbsp;OST\u0026alpha;/\u0026beta; at an overall resolution of 2.7\u0026nbsp;\u0026Aring; using 954,511 particles from the best class (Supplementary Fig. 1a \u0026amp; 2). In total, 9-328 residues of\u0026nbsp;OST\u0026alpha; and 18-64 residues of OST\u0026beta; were built into the EM density, except for some unstructured segments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe overall structure of OST\u0026alpha;/\u0026beta; displays a dimer of two heterodimers, which is consist with its size-exclusion chromatography behavior\u0026nbsp;(Supplementary Fig. 1b). Each protomer consists of one molecule of OST\u0026alpha; and one molecule of OST\u0026beta;, with OST\u0026alpha; in the center forming the symmetric interfaces\u0026nbsp;(Fig. 2a). OST\u0026alpha; adopts a seven-transmembrane (7-TM) helical architecture, with the helices arranged in a counterclockwise orientation when viewed from the extracellular side\u0026nbsp;(Supplementary Fig. 1d and e). These helices display characteristic tilts and bends that generate tight inter-helical packing reminiscent of class-A GPCRs\u003csup\u003e29\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe two OST\u0026alpha; molecules form a central lipid-filled cavity at the dimer interface within the membrane (Fig. 2b). This cavity accommodates multiple well-defined densities that could be modeled as cholesterol and phospholipids, which we tentatively fitted as phosphatidic acid (PA) based on the shape of the density. This central cavity is sealed on both the extracellular and intercellular sides. On the extracellular side, side chains from TM4, including Leu114, Leu196, Leu199 and Pro203 neighboring residues, completely occlude the opening (Fig. 2c). On the intracellular side, Pro82, Ile83, and Tyr326 form the constriction that close the other end of the cavity (Fig. 2d). We speculate that lipids within the cavity likely contribute to the tight dimerization by providing additional hydrophobic stabilization. The intracellular loop between TM3 and TM4 (Loop\u003csup\u003eTM3-4\u003c/sup\u003e penetrates partially into the membrane, and Cys160-164 and Cys166-167 are palmitoylated, further stabilizing membrane interactions.\u003c/p\u003e\n\u003cp\u003eThe N-terminal region of OST\u0026alpha; folds into three short \u0026alpha;-helices on the extracellular side, that directly engage the extracellular helix of OST\u0026beta; (Fig. 2e). OST\u0026beta; itself contains a single transmembrane helix that packs against TM1 and TM7 of OST\u0026alpha; forming a large interface of 1410\u0026nbsp;\u0026Aring;\u003csup\u003e2\u003c/sup\u003e between two proteins. This interface is strengthened by extensive hydrophobic contacts as well as three specific hydrogen bonds: Arg29 of OST\u0026beta; with Gly280 on TM7 of OST\u0026alpha;, Glu23 on the extracellular helix of OST\u0026beta; with Arg13 of the OST\u0026alpha; N-terminal loop, and Glu31 of OST\u0026beta; with Cys284 of OST\u0026alpha; TM7 (Fig. 2e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe structure of OST\u0026alpha;/\u0026beta; reveals architectural features that diverge substantially from canonical members of SLC superfamily (Supplementary Fig. 3). Most SLC transporters consist of a single polypeptide chain with a self-contained multi-helical transmembrane bundle, typically organized into symmetry-related repeats that enable conformational cycling for alternating access. In contrast, OST\u0026alpha;/\u0026beta; functions as a dimer of heterodimer, in which OST\u0026alpha; contributes a 7-TM bundle while OST\u0026beta; provides only a single transmembrane helix, forming a 1:1 complex protomer. Moreover, OST\u0026alpha; lacks the inverted repeat topology and rocker-switch or elevator-like domains characteristic of SLC families such as SLC6, SLC7, SLC17, and SLC25\u003csup\u003e30-33\u003c/sup\u003e. Instead, its 7-TM bundle resembles a GPCR-like helical architecture\u003csup\u003e29\u003c/sup\u003e rather than an SLC transporter fold\u003csup\u003e34-36\u003c/sup\u003e. Together, these features indicate that OST\u0026alpha;/\u0026beta; represents a topologically and mechanistically distinct transporter, employing a heterodimeric 7TM+1TM assembly and a lipid-stabilized dimeric interface unlike any previously characterized SLC topology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe TC-bound structure revealed a facilitated lateral diffusion mechanism for substrate translocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the transport mechanism of this unique member of SLCs, we solved the TC-bound OST\u0026alpha;/\u0026beta; structure by incubating the protein with 700 \u0026mu;M TC, at a resolution of 3.2 \u0026Aring; (Fig. 3a, Supplementary Fig. 4). Although processed without imposed symmetry (C1), two TC densities were observed symmetrically on opposite sides of OST\u0026alpha; at positions previously occupied by cholesterol molecule in the apo structure (Fig. 2b). The TC molecule occupies the surface-exposed groove between TM5 and TM6 of OST\u0026alpha;. Surface electrostatic analysis revealed a positively charged niche at the intracellular entrance of this groove, where Arg241 and Arg244 interact with the sulfonate moiety of TC via electrostatic interactions, including a salt bridge (Fig. 3b). In addition, Gln260 forms a hydrogen bond with the carbonyl group of TC. The TC molecule is further stabilized by hydrophobic interaction contributed mainly by the membrane-inserted Loop\u003csup\u003eTM3-4\u003c/sup\u003e and its palmitoylated segment, forming a semi-open pocket embedded within the lipid bilayer (Fig. 3b).\u003c/p\u003e\n\u003cp\u003eSuperposition of the TC-bound and apo structures yielded an RMSD of 0.663 \u0026Aring; over 699 residues aligned C\u0026alpha; atoms, showing minimal backbone rearrangement and arguing against classical conformational change-based mechanisms used by many SLC transporters, such as rocker-switch, elevator, or alternating-access modes\u003csup\u003e34-36\u003c/sup\u003e. Nevertheless, subtle but meaningful conformational changes are observed in residues that interact with\u0026nbsp;TC, particularly\u0026nbsp;near the entry site: the side chain of Arg241 rotates by 4.9 \u0026Aring; relative to its position in the apo structure, avoiding the steric clash while also keeping an\u0026nbsp;electrostatic interaction with\u0026nbsp;the sulfonate moiety of\u0026nbsp;TC (Fig. 3c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlong the lateral groove, several additional polar residues, such as Lys191, Asn222, Thr229, and Ser271, extend from the binding site toward the membrane and, together with several hydrophobic residues from TM5 and TM6, form an amphipathic diffusion path (Fig. 3d). Sequence alignment demonstrated that these residues, including those directly coordinating TC and those lining the putative diffusion path, are highly conserved among OST\u0026alpha; homologues (Fig. 3e). Consistently, transport assays using radiolabeled [\u0026sup3;H]-TC combined with site-directed mutagenesis revealed that mutations in the binding site (R241A and R244A) as well as residues along the proposed diffusion path (e.g., K191A) markedly reduced transport activity (Fig. 3f). In addition, deletion of the palmitoylated segment of Loop\u003csup\u003eTM3-4\u003c/sup\u003e showed a significant reduced transport activity, indicating its important role in consisting the\u0026nbsp;semi-open pocket embedded within the lipid bilayer.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the existence of this\u0026nbsp;diffusion path, we performed molecular dynamics (MD) simulations to estimate the energy required for a TC molecule to traverse either the central cavity or the lateral surface groove. The calculations revealed that TC passage through the lateral surface groove displays a much lower energy barrier than passage through the central cavity, which is highly energetically unfavorable\u0026nbsp;(Fig. 4a). Moreover, our transport activity assays with\u0026nbsp;site-directed mutagenesis also indicated that the central cavity does not function as a transport path (Supplementary Fig. 5).\u003c/p\u003e\n\u003cp\u003eTogether, our results strongly support a\u0026nbsp;facilitated lateral diffusion mechanism, in which bile acids diffuse across the membrane along the surface groove of OST\u0026alpha;/\u0026beta;, driven solely by their concentration gradient.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOST\u0026alpha;/\u0026beta; is the principal basolateral bile acid exporter in ileal enterocytes and a key determinant of enterohepatic bile acid circulation, yet its transport mechanism has remained unresolved despite decades of physiological, biochemical and clinical work. Here we provide high-resolution structures of human OST\u0026alpha;/\u0026beta; together with comprehensive functional analysis, and show that this heterodimeric SLC operates through an unexpected facilitated lateral diffusion mechanism. Rather than forming a classical translocation pore, OST\u0026alpha;/\u0026beta; uses a membrane-embedded, surface-exposed groove to mediate sodium- and ATP-independent, bidirectional diffusion of bile acids along their concentration gradient.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlternating-access, rocker-switch or elevator-type transporters typically rely on substantial rearrangements of transmembrane helices to alternately expose a central binding site to opposite sides of the membrane\u003csup\u003e34-36\u003c/sup\u003e. By contrast, superposition of our apo and TC-bound structures reveals minimal backbone rearrangement, with ligand binding accommodated largely by local side-chain adjustments around the entry site. The two OST\u0026alpha; subunits enclose a lipid-filled central cavity that is sealed on both extracellular and intracellular sides, structurally incompatible with a water-filled translocation pathway. Instead, TC binds in a lateral surface groove at the OST\u0026alpha; membrane interface, coordinated by conserved basic and polar residues and buttressed by a palmitoylated Loop\u003csup\u003eTM3-4\u003c/sup\u003e that shapes a semi-open pocket within the bilayer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, structural analysis combined with biochemical assays and MD simulation, we can depict the essential snapshots of how substrates are laterally diffused facilitated by OST\u0026alpha;/\u0026beta; (Fig. 4b, Supplementary Movie 1). In the physiological situation, bile acids were absorbed by ASBT in a sodium-dependent manner from the intestinal into enterocyte, then carried by IBABP towards the basolateral membrane where OST\u0026alpha;/\u0026beta; localized (Fig. 4c). As result, there is usually a higher concentration of bile acids than portal blood. Therefore, the facilitated diffusion should normally start from semi-open pocket within the bilayer, as we captured in the TC-bound structure (Snapshot 1). Along the amphiphilic groove towards the portal blood, conserved residues lead the flow of bile acids outwards, especially Lys191, as confirmed by our assays (Snapshot 2). Afterwards, bile acids would continue to move along the groove until it meets Loop\u003csup\u003eTM4-5\u003c/sup\u003e. According to the simulation, energies provided by the gradient is sufficient to expel the loop for the move on of bile acids (Snapshot 3). Mutational disruption of key residues in this groove strongly impairs diffusion, and MD simulations demonstrate that diffusion along this lateral pathway is energetically much more favorable than passage through the central cavity. Together, these observations support a model in which bile acids remain largely within the lipid phase and diffused laterally along an amphipathic surface groove, rather than being alternately buried and exposed in a central aqueous cavity.\u003c/p\u003e\n\u003cp\u003eBeyond establishing a mechanistic model for OST\u0026alpha;/\u0026beta; itself, our work has broader implications for the SLC superfamily and for membrane transport paradigms in general. OST\u0026alpha;/\u0026beta; possesses a GPCR-like 7-TM architecture and a dimer-of-heterodimers organization that are distinct from the major SLC folds described to date, and uses a membrane-embedded lateral groove rather than a deeply buried central cavity to translocate amphipathic substrates. OST\u0026beta; appears to act as a structural brace that locks the 7-TM core of OST\u0026alpha; into a competent conformation and helps organize the extracellular architecture, without directly contributing to the lateral transport groove.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the central position of OST\u0026alpha;/\u0026beta; at the interface between intestinal reabsorption and hepatic uptake, the structural framework presented here provides a rational basis for designing small molecules that modulate its activity, with potential applications in cholestatic liver disease, bile acid diarrhoea and metabolic disorders driven by altered bile acid signaling.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length human OST\u0026alpha; gene (UniProt ID: Q86UW1) and OST\u0026beta; gene (UniProt ID: Q86UW2) were synthesized by Sangon Biotech Company with codon optimization for eukaryotic expression. The OST\u0026alpha; was cloned into a pCAG vector with a C-terminal Flag tag (DYKDDDDK) using the ClonExpress\u0026reg; II One Step Cloning Kit (Vazyme Biotech Co., Ltd). The OST\u0026beta; was subcloned into a pCAG vector without any tag. Using homologous recombination of multiple fragments, all point mutations were introduced by designing forward and reverse primers with point mutations and overlapped amplifying fragments.\u003c/p\u003e\n\u003cp\u003eFor protein expression, HEK293F cells were cultured in SMM 293T-II medium (Sino Biological Inc.) at 37℃ and 130 rpm with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were transfected when the density reached 2.5~3.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mL. For cell transfection, 1.5 mg OST\u0026alpha; expression plasmid and 1.5 mg OST\u0026beta; expression plasmid were premixed with 6 mg linear polyethylenimine (PEI) (Polysciences, Inc) of 25,000 MW in 45 mL fresh medium for 15 min, then the mixture was added to 800 mL HEK293F cells, supplemented with another 45 mL medium, and incubated once more for 15 min at rest. The transfected cells were grown at 37℃ for 48-60 h before harvesting. Cell pellets were resuspended in the lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl (for the SPR experiment, the buffer system was adjusted to 50 mM HEPES-KOH pH 7.5, 150 mM NaCl, the same below) and 20% (v/v) glycerol after centrifugation at 5,000 rpm for 7 min. The suspension was flash-frozen in liquid nitrogen and stored at -80℃ for further use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor protein purification, the collected cells were lysed using an AH-1500 High Pressure Homogenizer (ATS inc.) with 5 passes at ~600 bar, followed by centrifugation at 17,300 \u0026times; g for 20 min to remove the cell debris. The supernatant was subjected to ultracentrifugation at 45,000 rpm for 45 min (Beckman, Type 70 Ti). The membrane precipitates were collected and incubated with the lysis buffer plus additional 1% (w/v) lauryl maltose neopentyl glycol (LMNG, NG310, Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS, C6013-25, Anatrace) at 6\u0026deg;C for 2 h. Insoluble material was removed by ultracentrifugation at 45,000 rpm for 45 min (Beckman, Type 70 Ti) at 4℃. The supernatant was incubated in a decolorization shaker with the anti-FLAG M2 affinity gel (Sigma) on ice for 1 h. The resin was then loaded onto the column and washed six times, each time with 5 mL of wash buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG. Protein was eluted in batches to 6 mL after 15 min incubation with elution buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG plus 200 \u0026mu;g/mL FLAG peptide. The eluate was then collected and concentrated using a 100-kDa MWCO Amicon Ultra centrifugal filter (Millipore) before being applied to size-exclusion chromatography by a Superose 6 Increase 10/300 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.005% (w/v) LMNG. Peak fractions were pooled and flash-frozen in liquid nitrogen for future functional experiments or concentrated for nanodiscs reconstitution. All steps were performed either in cold storage or on ice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanodisc preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor nanodisc preparation, the concentrated OST\u0026alpha;/\u0026beta; complex sample was reconstituted into lipid nanodiscs by mixing with purified MSP1D1 scaffold protein and porcine brain polar lipid extract (BPL, Avanti) at a 1:4:240 molar ratio. After 1 h of incubation on ice with shaking, 800 mg/mL Bio-Beads SM-2 resin (Bio-Rad) was added to the mixture to initiate the nanodiscs reconstitution reaction by removing the detergent. The mixture was incubated overnight in cold storage with constant rotation. The bio-beads were removed, and the mixture was cleared of debris by centrifugation. The supernatant was loaded onto a Superose 6 Increase 10/300 GL column in 50 mM HEPES-KOH pH 7.5, 150 mM NaCl buffer without detergent. Peak fractions were collected and concentrated for biochemical studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface plasmon resonance (SPR) experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll SPR experiments were performed on a Biacore 8000 instrument (Cytiva) at 25\u0026deg;C in buffer of 50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 5% DMSO. The purified OST\u0026alpha;/\u0026beta; in lipid nanodiscs was covalently immobilized onto the series S CM5 sensor chips (Cytiva) by amine-coupling chemistry. Different types and concentrations of bile acid in the running buffer were serially flowed over the chip surface with the immobilized protein and the blank for 1 min at a flowrate of 30 \u0026mu;L min\u003csup\u003e-1\u003c/sup\u003e. The data were fitted with steady-state affinity binding model using Biacore Insight Evaluation software. The graphs were plotted using OriginPro 2023 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell-based taurocholic acid transport assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild type or mutants of OST\u0026alpha;/\u0026beta; were subcloned into the pCAG vector, respectively. HEK293F cells at a density of 1.5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cells per mL were transfected with the empty vector or OST\u0026alpha;/\u0026beta; constructs. 24 h after transfection, cells were collected by centrifugation and resuspended for the Hank\u0026rsquo;s Balanced Salt Solution (137 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.81 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.49 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 4.17 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.34 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.44 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.44 mM D-Glucose). Cell counts were determined using Coulter counting and microscopic visualization. For all assay system, a 200 \u0026mu;L aliquot of cell suspension contains 3\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cells. For the [\u003csup\u003e3\u003c/sup\u003eH] taurocholic acid accumulation assay, cells were resuspended and incubated at 37\u0026deg;C with the HBSS buffer, containing 50 nM [\u003csup\u003e3\u003c/sup\u003eH] taurocholic acid (specific activity 20 Ci\u0026thinsp;mmol\u003csup\u003e\u0026minus;1\u003c/sup\u003e, American Radiolabeled Chemicals). The loading process was stopped by centrifugation at indicated time points. Cells were then washed twice with the ice-cold HBSS buffer with 5 \u0026mu;M taurocholic acid, and resuspended with the same buffer plus 1% Triton X-100 for cell lysis. The radioactivity in the cell lysis was counted using liquid scintillation counting (Tri-Carb 2910TR, PerkinElmer). For the taurocholic acid efflux assay, cells were first loaded in HBSS buffer, plus 50 nM [\u003csup\u003e3\u003c/sup\u003eH] taurocholic acid for 10 min, then washed and resuspended with [\u003csup\u003e3\u003c/sup\u003eH] taurocholic acid-free HBSS buffer. 200 \u0026mu;L aliquots were taken immediately after resuspension (defined as the zero time point) or at other indicated time points. Cells were centrifuged and washed twice with 1 mL ice-cold HBSS buffer, and resuspended with the same buffer plus 1% Triton X-100 for scintillation counting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM sample preparation and data acquisition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the wild-type sample, 3.5 \u0026mu;L of purified OST\u0026alpha;/\u0026beta; at a concentration of ~2 mg/mL was applied to glow-discharged holey carbon grids QUANTIFOIL (Au R2/1, 300-mesh,). The grids were blotted with filter paper for 3.5 s and zero blotting force. Then, the grids were plunged into liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (FEI) under 100% humidity at 8\u0026deg;C. A total of 9,788 micrograph stacks were automatically collected with EPU 2 software on a Titan Krios 300 kV transmission electron microscope equipped with a K3 Summit direct electron detector (Gatan) and a GIF Quantum energy filter (Gatan) at a defocus range of -2.3 to -1.3 \u0026mu;m with a magnification of \u0026times;105,000, resulting in a pixel size of 0.82 \u0026Aring;. Each movie stack containing 32 frames was exposed in a super-resolution mode, with a total dose of 55 e\u003csup\u003e-\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo obtain the structure of the sodium taurocholate bound OST\u0026alpha;/\u0026beta; complex, 500 \u0026mu;M TC was maintained in the buffer throughout the protein purification process. The purified protein concentrated to ~3 mg/ml was incubated with 200 \u0026mu;M sodium taurocholate on ice for 30 min. After that, aliquots of 3.5 \u0026mu;L protein complex were applied to glow-discharged holey carbon grids QUANTIFOIL (Au R2/1, 300-mesh,) The grids were blotted with filter paper with a 3.5 s blotting time and zero blotting force. Then the grids were plunged into liquid ethane cooled with liquid nitrogen using a Vitrobot Mark IV (FEI) under 100% humidity at 8\u0026deg;C. A total of 6,137 micrograph stacks were collected in the same manner as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDose-fractionated image stacks were applied to motion correction and contrast transfer function estimation, which are both implemented in cryoSPARC 4.7.1\u003csup\u003e37\u003c/sup\u003e. Particles were automatically picked with template picker or topaz pick and extracted for further 2D and 3D processing.\u003c/p\u003e\n\u003cp\u003eFor OST\u0026alpha;/\u0026beta;, first, 2D classification was performed using particles blob picked from the two datasets. The top 59,926 particles were selected as template to template picking, followed by multiple rounds of 2D classification. The selected 1,375,076 high-quality particles were subjected to ab-initio reconstruction, and the best class was chosen for homogeneous refinement and NU refinement. Subsequently, template picking and Topaz picking were conducted using all micrographs. After multiple rounds of 2D classification, ab-initio reconstruction and heterogeneous, 954,511 particles were further subjected to homogeneous refinement and NU refinement, yielding a reconstruction map at an average resolution of 2.68 \u0026Aring; (Fig. S2).\u003c/p\u003e\n\u003cp\u003eFor TC bound OST\u0026alpha;/\u0026beta;, the workflow is similar to the above. A total of 6,137 movies were used for calculation, and finally, 146,494 particles were further subjected to homogeneous refinement and NU refinement, yielding a reconstruction map at an average resolution of 3.20 \u0026Aring; (Fig. S3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe final local refinement map was used for model building in Coot. Using UCSF Chimera, the initial rigid body model of OST\u0026alpha;/\u0026beta; predicted by Alphafold 2\u003csup\u003e38\u003c/sup\u003e, was docked into the construction map. Then, model building and refinement were performed manually by Coot. After several rounds of manual refinement, the model was improved by iterative cycles of refinement using real_space_refine in Phenix\u003csup\u003e39,40\u003c/sup\u003e. The final model was validated by phenix.validation_cryoem, and all the figures were prepared with PyMOL (https://pymol.org) or ChimeraX\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWell-tempered meta-dynamics (WT-MTD) simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCHARMM-GUI\u003csup\u003e42-44\u003c/sup\u003e website was utilized to embed the protein, solvate the complex and generate the force field. The complexes were embedded into a bilayer consist of 70% palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) molecules with 30% cholesterol and solvate with the TIP3P model and 150 mM KCl at PH 7.0. Proteins of complexes were described by CHARMM36m forcefield, and the other ligands\u0026rsquo; parameters were generated by CGenFFin CHARMM-GUI\u003csup\u003e45,46\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAll of the MD simulations were performed with GROMACS 2025.2 at a constant temperature of 310 K. Each of simulations was started with energy-minimized, and then run 750 ps NVT equilibration steps with positional restraints. Next, 100 ns NVT unrestrained production simulation were operated. The equilibration of the systems was confirmed through monitoring time-dependent RMSD of the complexes. The protein-ligand complex, membrane, and solvent were coupled separately to a temperature bath of 303.15 K using the velocity-rescaling thermostat with a relaxation time of 1.0 ps. All covalent bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing stable integration at the chosen time step. Van der Waals interactions were treated using a force-switch cutoff scheme with switching starting at 1.0 nm and a cutoff at 1.2 nm. The neighbor list was updated every 20 steps using the Verlet cutoff scheme. Electrostatic interactions were computed using the particle mesh Ewald (PME) method with a real-space cutoff of 1.2 nm.\u003c/p\u003e\n\u003cp\u003eSteered Molecular Dynamics (SMD) simulations were performed using the PLUMED plugin. The reaction coordinate (Collective Variable, CV) was defined as the z-component of the center of mass position of the TCH molecule. Moving restraint was applied with a velocity of 1 \u0026Aring;\u0026middot;ns\u003csup\u003e-1\u003c/sup\u003e and a moving force constant k from 0 to 200 kJ\u0026middot;mol\u003csup\u003e-1\u003c/sup\u003e\u0026middot;nm\u003csup\u003e-2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWT-MTD production simulations\u003csup\u003e47\u003c/sup\u003e were using the same conditions applied during equilibration. An integration time step of 4 fs was employed. Two collective variables (CVs) were chosen: the z-component of the center of mass of the TCH molecule (excluding hydrogens) and the Euclidean distance between S of TCH and N of Lys191\u0026rsquo;s side chain. A well-tempered bias was applied with Gaussian widths of 0.10 nm (for both CVs), an initial height of 2.0 kJ\u0026middot;mol⁻\u0026sup1;, and a bias factor of 15. Harmonic upper and lower walls were applied to restrict the sampling space to the physically relevant region.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Yong-Xiang Gao at the Center for Integrative Imaging, University of Science and Technology of China during cryo-EM image acquisition. This work was supported by the Ministry of Science and Technology of China (2024YFA1307900 to W.-T.H, 2024YFA1306103 to Y.C) and the Ministry of Science and Technology of China (2024YFA1306103 to Y.C), National Natural Science Foundation of China (32371257 to Y.C) and USTC Research Funds of the Double First-Class Initiative (YD9100002057 to W.-T.H),\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.X., Y.C. and W.-T.H. conceptualized this study. C.-Z.Z and Y.C. supervised the project. K.X., and W.-T.H. designed all the experiments. K.X., C.-Y.C. and G.-T.C. performed cloning. K.X., and F.-F.W. performed biochemical characterization. K.X. performed expression, purification, cryo-EM sample preparation, screening, cryo-EM data collection and model refinement. K.X. and G.H. performed structure determination. K.X. and B.-R.L. performed MD simulations. K.X., J.W. and B.W. performed data analysis. K.X., W.-T.H., C.-Z.Z., and Y.C. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cryo-EM density maps of two structures have been deposited at the Electron Microscopy Data Bank under accession codes: EMD-XXXX for OST\u0026alpha;/\u0026beta;, EMD-XXXX for TC-bound OST\u0026alpha;/\u0026beta;, and the coordinates have been deposited at the Protein Data Bank under accession codes: XXXX for OST\u0026alpha;/\u0026beta; and XXXX for TC-bound OST\u0026alpha;/\u0026beta;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDawson, P.A. et al. 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Exploring the free energy surfaces of clusters using reconnaissance metadynamics. \u003cem\u003eJ Chem Phys\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, 114109 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8254570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8254570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOSTα/β is the essential basolateral transporter responsible for returning reabsorbed bile acids to the portal circulation, yet its molecular mechanism remains unclear. Here, we report the cryo-electron microscopy structure of human OSTα/β at 2.7 Å resolution, revealing a GPCR-like 7-TM architecture distinct from all established SLC transporter folds. We further determined the taurocholate (TC)-bound structure at 3.2 Å, showing that the substrate laterally binds to a surface groove on OSTα, but not in the central cavity. The TC molecule is captured by a line of conserved polar residues along the groove, which were further proved by structure-guided mutagenesis combined with transport assays. Molecular dynamics simulations also suggest that it is energetically favored for TC moving along the surface groove. Together, structural analysis, functional assays, and simulations demonstrate that OSTα/β mediates bile acid transmembrane translocation via a facilitated lateral diffusion mechanism with subtle conformational change, which is distinct from known SLCs. These findings establish a mechanistic framework for OSTα/β function in enterohepatic bile acid circulation and provide a foundation for therapeutic modulation of bile acid homeostasis.\u003c/p\u003e","manuscriptTitle":"Structural basis of bile acid lateral diffusion facilitated by the OSTα/β transporter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 09:34:21","doi":"10.21203/rs.3.rs-8254570/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5350557e-72d6-4c69-8503-bfbccc5237b1","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61009421,"name":"Biological sciences/Biochemistry/Proteins/Membrane proteins"},{"id":61009422,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2026-03-02T18:50:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 09:34:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8254570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8254570","identity":"rs-8254570","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}