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Structures of Ostα/β reveal a unique fold and bile acid transport mechanism

Nature volume 651, pages 260–267 (2026)Cite this article

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Abstract

Bile acid and steroid hormone homeostasis are critical for human health, with disruptions linked to metabolic and endocrine disorders1,2. The organic solute transporter Ostα/β, essential for bile acid efflux in enterohepatic circulation3, has long defied mechanistic elucidation. Here we present cryogenic electron microscopy structures of human Ostα/β in apo and substrate-bound states at 2.6–3.1 Å resolution, revealing a distinctive membrane protein architecture that defines a new transporter class. Ostα/β forms a symmetric tetramer of heterodimers, with each Ostα subunit showing a new seven-transmembrane fold, augmented by a single transmembrane helix of Ostβ. This architecture is stabilized by extensive lipid modifications, including a palmitoylated cysteine-rich motif that forms a lateral substrate-binding groove. The structures uncover a unique transport pathway featuring two substrate-binding sites connected by an amphipathic helix-gated conduit. This design, conserved in the evolutionarily related TMEM184 family, suggests an ancient mechanism for substrate translocation. Electrophysiological studies demonstrate voltage-sensitive, bidirectional transport driven by electrochemical gradients, elucidating the efflux role of Ostα/β in vivo. Lipid interactions, notably palmitoylation-dependent trafficking, emerge as critical for stability and function. These findings clarify the molecular mechanism of Ostα/β, provide a structural basis for disease-associated mutations4,5 and establish a paradigm for lipid-modified membrane transport.

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Fig. 1: Structure of dimerized heterodimer of human Ostα/β.
Fig. 2: Substrate-binding groove of Ostα/β.
Fig. 3: Solvent-exposed tunnel in the extracellular side.
Fig. 4: Voltage-dependent transport Ostα/β.
Fig. 5: Key interactions mediating dimer–heterodimer formation.

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Data availability

The coding sequences for human wild-type Ostα (UniProt Q86UW1) and Ostβ (UniProt Q86UW2) are available. The density maps and structure coordinates have been deposited to the Electron Microscopy Data Bank and the PDB under accession numbers EMD-64370 and 9UO2 (Ostα/β-apo complex); EMD-64364 and 9UNV (Ostα/β–TLCA complex); EMD-64369 and 9UO1 (Ostα/β–DHEAS complex). For MD simulations, the initial coordinate and simulation input files and a coordinate file of the final output are available at Zenodo (https://doi.org/10.5281/zenodo.17656875)57. Bioinformatic data and classification information were obtained from publicly available sources: TMEM184 family sequences from the National Center for Biotechnology Information, TMEM184A NP_001091089.1, TMEM184B NP_001182000.1 and TMEM184C NP_060711.2, TCDB classifications from the CDB through http://www.tcdb.org, accessed through 2.A.82.1, and domain annotations from Pfam at https://www.ebi.ac.uk/interpro/ (InterPro entry IPR005178). Foldseek is available through https://doi.org/10.1038/s41587-023-01773-0 and was used for sequence similarity searches. All other data supporting the findings of this study are available within the Article and its Supplementary Information. Any extra materials are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The cryo-EM data were collected at the Advanced Center for Electron Microscopy, Shanghai Institute of Materia Medica (SIMM). We thank all staff, especially W. Hu, K. Wu and S. Li at the institution, for their assistance in cryo-EM data collection. Thank you to J. Li for the generous advice on data processing. We express our appreciation for the provision of experimental instruments from the Experimental Nuclear Medicine Laboratory, Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine. We express our appreciation to H. Song for providing experimental instruments. This work was supported by the National Natural Science Foundation of China (82495184, 32130022, 82121005 to H.E.X.; 82130018 to X.M.; 32301016 to C.W.), the National Key R&D Program of China (2022YFA1302900 to W.Y., 2022YFC2703105 to H.E.X.), National Key R&D Program “Strategic Scientific and Technological Innovation Cooperation” Key Project (2022YFE0203600) released by the Ministry of Science and Technology, CAS Strategic Priority Research Program (XDB37030103 to H.E.X.), Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.), Shanghai Municipal Science and Technology Major Project (H.E.X.) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0830000 to H.E.X.).

Author information

Author notes

  1. These authors contributed equally: Xuemei Yang, Nana Cui, Tianyu Li, Xinheng He

Authors and Affiliations

  1. Research Center for Medicinal Structural Biology, National Research Center for Translational Medicine at Shanghai, State Key Laboratory of Medical Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Xuemei Yang, Canrong Wu & H. Eric Xu

  2. Division of Gastroenterology and Hepatology, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, State Key Laboratory for Oncogenes and Related Genes, NHC Key Laboratory of Digestive Diseases, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Institute of Digestive Disease, Shanghai, China

    Nana Cui & Xiong Ma

  3. The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Tianyu Li, Xinheng He, Heng Zhang, Yang Li & H. Eric Xu

  4. University of Chinese Academy of Sciences, Beijing, China

    Tianyu Li, Xinheng He & Yang Li

  5. Institute of Aging and Tissue Regeneration, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Xiong Ma

  6. The Shanghai Advanced Electron Microscope Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    H. Eric Xu

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Contributions

X.Y. designed the expression constructs, purified the Ostα/β protein samples, prepared cryo-EM grids, calculated cryo-EM data, built and refined structural models, and prepared figures and wrote the initial article draft. N.C. conducted the [3H]TCA transporter uptake assay, flow cytometry and immunofluorescence analysis, co-immunoprecipitation and prepared corresponding figures and methods supervised by X.M. T.L. conducted the electrophysiological recordings and prepared corresponding methods and figures, supervised by Y.L. X.H. performed the molecular dynamics simulations and prepared corresponding methods, figures and videos. H.Z. participated in the discussion on experiment design and structural analysis. C.W. participated in protein purification and sample preparation. H.E.X., in collaboration with X.M., supervised the project. H.E.X. modified the paper.

Corresponding authors

Correspondence to Xiong Ma or H. Eric Xu.

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Extended data figures and tables

Extended Data Fig. 1 Palmitoylation influences Ostα subcellular localization.

a Subcellular localization of Ostα (green), Ostβ-mCherry (red), plasma membrane (orange) and nuclei (blue). Scale bar = 10 μm. Results are representative of three independent experiments. b Plasma membrane Ostα expression in WT and mutants quantified by flow cytometry (FACS). Data are mean ± s.e.m. (n = 4 biological replicates per group, ****P < 0.0001). c Co-immunoprecipitation (IP) analysis of overexpressed Ostβ and Ostα mutants in HEK293T cells. Results are representative of two independent experiments. d Quantification of total Ostα expression levels assessed by FACS in WT and mutants. Data are mean ± s.e.m. (n = 3 biological replicates per group). The P values for comparisons between Ostα/β and the respective conditions were 0.9936, 0.4073, 0.3491, and 0.9703. e Inhibition of Ostα/β transport activity by 2-BP. [³H]-TCA uptake measured after 2-BP treatment. Data are mean ± s.e.m. (n = 6 biological replicates per group, **P = 0.0015; ****P < 0.0001). f Plasma membrane Ostα expression levels assessed by FACS in WT and 2-BP treatment, normalized to untreated WT Ostα/β. Data are mean ± s.e.m. (n = 3 biological replicates per group, ***P = 0.0009; ****P < 0.0001). g Dose-dependent degradation of Ostα in response to 2-BP treatment. β-actin and Na+/K+ ATPase served as controls. h Total Ostα expression levels assessed by FACS in WT and 2-BP treatment, normalized to untreated WT Ostα/β. Data are mean ± s.e.m. (n = 3 biological replicates per group, ****P < 0.0001). b, d-f, h Results are representative of three independent experiments. Data analyzed by one-way ANOVA with Fisher’s LSD test.

Source Data

Extended Data Fig. 2 Cholic acids binding site in different transporters.

a-b Surface representation of Ostα/βTLCA structure. The TLCA in magenta are shown in ball-stick model. c Hydrophilic surface of Ostα/βTLCA, same view as (b). d Inward conformation comparison between NTCP (PDB: 7ZYI) and ASBTnm (PDB: 3ZUX). e Surface representation of NTCP. Two glyco-chenodeoxycholic acid (GCDC) are coloured in yellow in ball-stick model. Cholesterol is coloured in light gray. f Hydrophilic surface of NTCP. g Surface representation of ASBTnm. Taurocholate is coloured in yellow in ball-stick model. Lipids are coloured in light gray in stick model. h Hydrophilic surface of ASBTnm.

Extended Data Fig. 3 Comparison of 3D maps and models refined with C1 and C2 symmetry.

Density maps and their corresponding atomic models were independently refined under C1 and C2 symmetry. The C2 maps are shown in gray with 50% transparency. C1 maps were generated de novo from the same dataset. All maps are visualized at a contour level of 0.35 with dust removal threshold set to 5. Subtle local differences between the two reconstructions are indicated by red arrows.

Extended Data Fig. 4 Structure alignment and ICH domain interaction with surrounding environment.

a Overall structure alignment among Ostα/βapo (blue), Ostα/βTLCA (violet) and Ostα/βDHEAS (green). The RMSD of Ostα/βTLCA and Ostα/βDHEAS compared with Ostα/βapo is 0.264 Å and 0.204 Å, correspondingly. b Lateral view from the extracellular side of substrate binding site. Surface is coloured based on Ostα/βapo structure hydrophobic properties. c The amphiphilic helix ICH is located between TM4 and TM5.

Extended Data Fig. 5 Sequence alignment of Ostα/β from different species.

Secondary structure assignments are based on the resolved Ostα (a) and Ostβ (b). Hom.s, Homo sapiens, Mus.m, Mus musculus, Mon.m, Monodon monoceros, Rou.a, Rousettus aegyptiacus, Dan.r, Danio rerio, Aha.p, Ahaetulla prasine, Gal.g, Gallus gallus, Dan.r, Danio rerio, Dro.m, Drosophila melanogaster, Cae.e, Caenorhabditis elegans.

Extended Data Fig. 6 Inner face key residues at the potential extracellular binding site.

a Hydrophilic surface of inner lateral lobe. b Hydrophilic surface of inner main lobe. Effects of mutants at the inner cavity on Ostα/β transport activity. c, d Ligands interaction with Ostα/β near the extracellular side. LigPlot+ analysis for cholesterol (blue, c) and DHEAS (green, d). Hydrogen bonds were defined using a donor–acceptor distance cutoff of 3.9 Å, and hydrophobic (non-bonded) contacts were identified within a 2.2–3.9 Å range. e MD simulation distance traces from Ostα/βDHEAS tetramer structure: Lig–G279 (DHEAS sulfur–G279 Cα), Lig–K191 (DHEAS sulfur–K191 side chain nitrogen), P209–E116 (minimum distance), and Lig–palm (minimum distance DHEAS sulfur-7 palmitoylated cysteines).

Extended Data Fig. 7 Lipids binding at the Ostα-Ostα’ interface.

a Head group of PE -like lipids located at the interface with positive potential. The surface electrostatic potentials were calculated by APBS. b Local densities of PE-like lipids in C2 Ostα/βapo map (contour level 0.2).

Extended Data Fig. 8 Cryo-EM structure of Ostα/βapo purified in the absence of CHS.

a, Cryo-EM map of Ostα/βapo purified without CHS, coloured by local resolution. The previously determined Ostα/βapo model (protein shown in green, four cholesterol molecules in yellow) was rigid-body fitted into the map for comparison (contour level 0.28). b, Fourier shell correlation (FSC) curve of the final reconstruction. c, Enlarged views of sterol-like densities observed at three sites, consistent with cholesterol binding. d, Angular distribution of particle orientations contributing to the reconstruction.

Extended Data Fig. 9 Representative lipid-like densities in the Ostα/βapo structure.

a Representative non-protein densities surrounding the Ostα/βapo structure (contour = 0.21). Putative cholesterols (excluding those at the substrate binding sites) shown in transparent gray, phospholipid-like densities in transparent purple. b Enlarged view of density resembling lysophosphatidylglycerol (LPG). c Enlarged view of density resembling PE. d Lipid-Ostα/β interaction interface 1 (IF1) with surrounding residues with extended hydrophobic side chains. e-g, Cholesterol-Ostα/β interaction interface 2-5 (IF2-5), with surrounding residues with extended hydrophobic side chains. h, [³H]-TCA uptake activity of IF mutants compared to WT Ostα/β. Data are mean ± s.e.m. (n = 6 biological replicates per group, ***P = 0.0001; ****P < 0.0001. i, FASC analysis of IFs mutants showing differential effects on Ostα cell surface expression. Data are mean ± s.e.m. (n = 3 biological replicates per group, ****P < 0.0001, ns = not significant P > 0.05, **P < 0.01. The exact P-values for the respective IF-mutants 1-5: 0.3014, 0.1436, 0.0067, 0.6246, >0.999). j, Quantification of plasma membrane (green) and total (gray) Ostα expression levels in WT and IF-mutants backgrounds, normalized to WT Ostα/β membrane expression. Data are mean ± s.e.m. (n = 3 biological replicates per group). Unpaired t-tests were used for statistical comparisons; “nd” indicates no significant difference. The exact P values were: >0.9999, 0.0194, 0.7840, 0.6885, 0.0233, and 0.4872. h, i, Data analyzed by one-way ANOVA with Fisher’s LSD test. h-j, Results are representative of three independent experiments.

Source Data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Information (download PDF )

This file contains: Supplementary Information 1 (Figs 1–11 and Tables 1 and 2); Supplementary Information 2 (raw SDS–PAGE and western blot source data for Extended Data Fig. 1c) and Supplementary Information 3 (chemical structures of ligands in the figures).

Reporting Summary (download PDF )

Supplementary Video 1 (download MP4 )

DHEAS upward movement in the tetrameric Ostα/β complex, trajectory 2. Time-evolution video from MD simulations of the tetrameric Ostα/β complex (trajectory 2), illustrating the upward movement of DHEAS during the production phase.

Supplementary Video 2 (download MP4 )

DHEAS upward movement in the tetrameric Ostα/β complex, trajectory 5. Time-evolution video from MD simulations of the tetrameric Ostα/β complex (trajectory 5), showing the upward movement of DHEAS during the production phase.

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DHEAS upward movement in the heterodimeric Ostα/β complex, trajectory 1. Time-evolution video from MD simulations of the heterodimeric Ostα/β complex (trajectory 1), depicting the upward movement of DHEAS during the production phase.

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Yang, X., Cui, N., Li, T. et al. Structures of Ostα/β reveal a unique fold and bile acid transport mechanism. Nature 651, 260–267 (2026). https://doi.org/10.1038/s41586-025-10029-7

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