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Structure of the bile acid transporter and HBV receptor NTCP

Nature volume 606, pages 1021–1026 (2022)Cite this article

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Abstract

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus–transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

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Fig. 1: Structure of mammalian NTCP.
Fig. 2: Substrate transport pathway of NTCP.
Fig. 3: Interactions between NTCP and preS1.
Fig. 4: Model of the mode of preS1 binding.

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Data availability

Cryo-EM maps and structure coordinates have been deposited in the Electron Microscopy Data Bank and the PDB, respectively, under the accession codes EMD-31837 and 7VAD (human NTCP(Q261A)–Fab); EMD-31838 and 7VAE (bovine NTCP(Q261A)–Fab); EMD-31839 and 7VAF (rat NTCP(Q261A)–Fab); EMD-31840 and 7VAG (human NTCP–Fab–myr-preS1); and EMD-32759 and 7WSI (human NTCP(WT)–Fab).

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Acknowledgements

We thank A. Tsutsumi, H. Yanagisawa, Y. Kise, Y. Sakamaki, M. Kikkawa, Y. Sugita and D. Im for their help in cryo-EM data collection, T. Tomita for allowing us to use the ultracentrifuge, and S.-Y. Park for discussions. Preliminary cryo-EM screening for antibody as a fiducial marker was performed using a Glacios at the RIKEN SPring-8 Center. This work was supported by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (grant numbers 19H03164 (U.O.), 19H00976 (T.S.), 18K05334 (N.N.) and 19H00923 (S.I. and N.N.)). This work is partially supported by the Knut and Alice Wallenberg foundation (D.D.), the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from Japan Agency for Medical Research and Development (AMED) under grant numbers JP19am0101115 (support no. 1570, 1846, 1848) and JP21am0101079 (S.I. and N.N.), an AMED Research Program on Emerging and Re-emerging Infectious Disease (20fk0108270h0001) (T.N.), the JST Core Research for Evolutional Science and Technology (JPMJCR20HA) (T.N.), JSPS Core-to-Core Program A (T.N.), the Joint Research Project of the Institute of Medical Science, the University of Tokyo (T.N.) and the Joint Usage/Research Center Program of Institute for Frontier Life and Medical Sciences, Kyoto University (N.N. and T.N.)

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Author notes

  1. These authors contributed equally: Jinta Asami, Kanako Terakado Kimura, Yoko Fujita-Fujiharu

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Jinta Asami, Hanako Ishida, Zhikuan Zhang, Toshiyuki Shimizu & Umeharu Ohto

  2. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Kanako Terakado Kimura, Yayoi Nomura, Kehong Liu, Tomoko Uemura, Yumi Sato, Masatsugu Ono, So Iwata & Norimichi Nomura

  3. Laboratory of Ultrastructural Virology, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan

    Yoko Fujita-Fujiharu & Takeshi Noda

  4. Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Yoko Fujita-Fujiharu & Takeshi Noda

  5. CREST, Japan Science and Technology Agency, Kawaguchi, Japan

    Yoko Fujita-Fujiharu & Takeshi Noda

  6. RIKEN SPring-8 Center, Sayo-gun, Japan

    Masaki Yamamoto, Hideki Shigematsu & So Iwata

  7. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    David Drew

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  1. Jinta Asami
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Contributions

J.A., N.N. and U.O. designed the experiments. J.A., H.I., Y.N., Y.S., M.O., D.D., N.N. and U.O. prepared recombinant proteins. K.L., T.U., S.I. and N.N. generated antibodies. J.A., K.T.K., Y.F.-F., Z.Z., M.Y., T.N., H.S., N.N. and U.O. performed cryo-EM analyses. J.A. and H.I performed the in vitro preS1 binding assay. K.T.K. performed the transport assay. J.A., D.D., N.N. and U.O. wrote the paper with assistance from all of the authors. S.I., T.S., N.N. and U.O. supervised the project.

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Correspondence to Norimichi Nomura or Umeharu Ohto.

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Extended data figures and tables

Extended Data Fig. 1 Sequence alignment of NTCP and ASBT.

Sequence alignment of human, bovine, and rat NTCPs and human ASBT, as well as bacterial homologues from Yersinia frederiksenii (ASBTYf) and Neisseria meningitidis (ASBTNM), were calculated using Clustal Omega. Coloured bars indicate locations of the transmembrane helices in the human NTCP. Residues involved in myr-preS1 binding are indicated by red boxes. Residues involved in Na binding (Na1 and Na2 sites) are highlighted in cyan and orange, respectively. Residues near the unmodelled lipid densities in the human NTCP–Fab structure and near the TCA molecule in the ASBTNM structure are highlighted in pink and yellow, respectively. Residues forming TM1 in bASBTs are boxed with black lines.

Extended Data Fig. 2 Sample preparation of mammalian NTCP.

a–c, Representative SEC of human (a), bovine (b), and rat (c) NTCP(Q261A). Absorbance profiles (top) and SDS–PAGE analysis of the peak fractions stained with CBB (bottom) are shown. Pooled fractions are shown as bars. Absorbances at 280 nm and 260 nm are shown as solid and dashed lines, respectively. Purifications were performed four, one, and four times for human, bovine, and rat NTCP, respectively. Uncropped blots are shown in Supplementary Fig. 1.

Extended Data Fig. 3 Cryo-EM analysis of mammalian NTCP–Fab complexes.

a–d, Data processing workflow of cryo-EM analysis of human (a), bovine (b), and rat (c) NTCP–YN69202Fab complexes and human NTCP–YN69202Fab complex with myr-preS1 (d). Representative motion-corrected micrograph (out of 5,434 (a), 2,712 (b), 9,932 (c), and 7,496 (d) total micrographs), 2D class averages, 3D class averages, gold-standard FSC curves of the final 3D reconstruction (resolution cut-off at FSC = 0.143), and the final 3D map (coloured according to the local resolution) are shown. 2D class averages were calculated using refined particles that were used for the final reconstruction. 3D classes selected for the following analyses are indicated with red dotted boxes.

Extended Data Fig. 4 Cryo-EM density maps of mammalian NTCPs in complex with Fab.

a, Overall structures of the human (left), bovine (middle), and rat (right) NTCP–YN69202Fab complexes. Cryo-EM maps (top) and ribbon models (bottom) are shown. NTCP and heavy and light chains of Fab are shown in green, cyan, and blue, respectively. b, Cryo-EM density maps of the rat NTCP–YN69202Fab complex around each TM helix of NTCP are shown.

Extended Data Fig. 5 Structural comparison between mammalian NTCP and bASBTs.

a, Structural comparison of human NTCP(Q261A), ASBTYf(E254A) (PDB ID: 4N7X, outward-open), wild-type ASBTYf (PDB ID: 4N7W, inward-open), and wild-type ASBTNM (PDB ID: 3ZUX, inward-open). Each structure was viewed from the extracellular side (top) and parallel to the membrane (bottom). RMSD values of each ASBT structure against the human NTCP structure are indicated (top). b, Structural comparison of the core (left) and panel (right) domains of NTCP and ASBT. Structures of human NTCP(Q261A) (light green), ASBTYf(E254A) (orange), wild-type ASBTYf (cyan), and wild-type ASBTNM (blue) are superposed for each domain. RMSD values of the ASBT structures against the human NTCP structure are indicated (top). c, Close-up view of sodium-binding sites. Na1 (top) and Na2 (bottom) binding sites in the structure of ASBTNM (left) and the corresponding sites of human NTCP (right) are shown. Side chains of residues involved in Na+ binding are labelled and shown as stick representations.

Extended Data Fig. 6 Sodium-dependent transport of NTCP constructs used in this study.

Michaelis–Menten transport kinetics of human NTCP wild type (circle)- and Q261A mutant (triangle)-mediated [3H]-TCA uptake. The specific uptake was calculated by subtracting the sodium-independent and endogenous TCA transport activities from the total uptake, as detailed in Methods. Data represent mean ± standard deviation of three independent measurements. The observed Michaelis constant, Km, for [3H]-TCA is ~9.5 µM (wild type). Estimation of accurate Km value of the Q261A mutant for [3H]-TCA is technically impossible owing to the low transport activity. The Vmax value of the Q261A-mediated uptake was 349.2 ± 103.3 pmol min−1 mg−1, which is fivefold lower than the corresponding value for wild type (1791 ± 154.7 pmol min−1 mg−1).

Source data

Extended Data Fig. 7 Structural comparison between wild-type and Q261A-mutant human NTCP.

a, Representative SEC of human NTCP (WT). Absorbance profiles (top) and SDS–PAGE analysis of the peak fractions stained with CBB (bottom) are shown. Pooled fractions are shown as bars. Absorbances at 280 nm and 260 nm are shown as solid and dashed lines, respectively. Purifications were performed nine times with similar results. The uncropped blot is shown in Supplementary Fig. 1. b, Data processing workflow of cryo-EM analysis of human NTCP (wild type)-YN69202Fab complex. Representative motion-corrected micrograph, 2D class averages, 3D class averages, gold-standard FSC curves of the final 3D reconstruction (resolution cut-off at FSC = 0.143), and the final 3D map (coloured according to the local resolution) are shown. 2D class averages were calculated using refined particles that were used for the final reconstruction. 3D classes selected for the following analyses are indicated with red dotted boxes. c, Structural comparison of wild-type and Q261A-mutant human NTCP. Structures of human NTCP (wild type) (grey) and human NTCP(Q261A) (light green) are superposed (upper left). Na1 (upper right) and Na2 (bottom right) binding sites in the structure of human NTCP (wild type) and human NTCP(Q261A) are shown. Side chains of residues involved in Na+ binding are labelled and shown as stick representations. Close-up view of human NTCP (wild type) marked with a black rectangle is shown (bottom left). The two observed densities are indicated in orange. Side chains of residues near the densities are shown as stick representations. Crossover regions of TM3 and TM8 is indicated by a black dotted circle.

Extended Data Fig. 8 NTCP–preS1 binding interface.

a, Topology diagrams of HBV envelope glycoproteins LHBs. b, Sequences of preS1 mutants used in Fig. 3c. Sequence of wild-type preS1 (2–48) and the residue numbers are indicated (top). Dots in the sequence indicate the same residues as the wild type. c, Structural comparison of predicted preS1-binding sites, patch 1 (left), and patch 2 (right), among mammalian NTCPs. Superposition of human (light green), bovine (cyan), and rat (magenta) NTCPs with patch 1 (in TM2–3 loop) and patch 2 (in TM5), indicated with rectangles (top). A detailed view and sequence of each site are shown (bottom).

Extended Data Fig. 9 Cryo-EM densities in the tunnel in NTCP.

Cryo-EM density maps for the human NTCP–YN69202Fab complex in the presence (green) and absence (orange) of myr-preS1. Only the densities in the channel are displayed.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Asami, J., Kimura, K.T., Fujita-Fujiharu, Y. et al. Structure of the bile acid transporter and HBV receptor NTCP. Nature 606, 1021–1026 (2022). https://doi.org/10.1038/s41586-022-04845-4

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