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Transport mechanism of presynaptic high-affinity choline uptake by CHT1

Nature Structural & Molecular Biology volume 31, pages 701–709 (2024)Cite this article

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Abstract

Choline is a vital nutrient and a precursor for the biosynthesis of essential metabolites, including acetylcholine (ACh), that play a central role in fetal development, especially in the brain. In cholinergic neurons, the high-affinity choline transporter (CHT1) provides an extraordinarily efficient reuptake mechanism to reutilize choline derived from intrasynaptical ACh hydrolysis and maintain ACh synthesis in the presynapse. Here, we determined structures of human CHT1 in three discrete states: the outward-facing state bound with the competitive inhibitor hemicholinium-3 (HC-3); the inward-facing occluded state bound with the substrate choline; and the inward-facing apo open state. Our structures and functional characterizations elucidate how the inhibitor and substrate are recognized. Moreover, our findings shed light on conformational changes when transitioning from an outward-facing to an inward-facing state and establish a framework for understanding the transport cycle, which relies on the stabilization of the outward-facing state by a short intracellular helix, IH1.

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Fig. 1: Architecture of human CHT1.
Fig. 2: Human CHT1 in the HC-3-bound outward-facing state.
Fig. 3: CHT1 in the inward-facing apo and choline-bound inward-facing occluded states.
Fig. 4: Conformational changes between CHT1HC-3/OF and CHT1choline/occ.
Fig. 5: Molecular mechanism of choline transport.

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Data availability

The cryo-EM density maps of the dimeric CHT1HC-3/OF, monomeric CHT1HC-3/OF, CHT1apo/IF and CHT1choline/occ have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession codes EMD-36025, EMD-36027, EMD-36029 and EMD-36030, respectively. The unsharpened cryo-EM maps, used for model building of the C-terminal region, have also been deposited in the EMDB as additional EM maps within the corresponding deposition entries. The coordinates have been deposited in the Protein Data Bank (PDB) under PDB IDs 8J74, 8J75, 8J76 and 8J77. Molecular dynamics trajectories were deposited into a public repository and are accessible at the following link: https://doi.org/10.5281/zenodo.10526461.

Source data are provided with this paper.

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Acknowledgements

We thank L. Chen (Peking University) and Y. Niu (Peking University) for their helpful discussions. We thank X. Huang, B. Zhu, X. Li and other staff members at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science for their support in cryo-EM data collection; and H. Zhang (Institute of Biophysics, Chinese Academy of Sciences) and T. Sun (Institute of Microbiology, Chinese Academy of Sciences) for their assistance in the [3H]choline-uptake assays. We thank Y. Ren (Institute of Biophysics, Chinese Academy of Sciences) for his assistance in the immunoblotting assays. We thank Y. Wu for his research assistance. This work is funded by Chinese National Programs for Brain Science and Brain-like Intelligence Technology (grant no. 2022ZD0205800 to Y.Z.), the National Key Research and Development Program of China (grant no. 2021YFA1301501 to Y.Z.), the National Natural Science Foundation of China (grant no. 92157102 to Y.Z.) and the Chinese Academy of Sciences Strategic Priority Research Program (grant no. XDB37030304 to Y.Z.).

Author information

Author notes

  1. These authors contributed equally: Yunlong Qiu, Yiwei Gao.

Authors and Affiliations

  1. Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yunlong Qiu, Yiwei Gao, Qinru Bai & Yan Zhao

  2. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Yunlong Qiu, Yiwei Gao, Qinru Bai & Yan Zhao

  3. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Yunlong Qiu, Yiwei Gao, Qinru Bai & Yan Zhao

  4. Beijing StoneWise Technology Co Ltd., Beijing, China

    Bo Huang

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Contributions

Y.Z. conceived the project and supervised the research. Y.Q. carried out molecular cloning experiments, expressed and purified protein samples and prepared samples for cryo-EM study. Y.G. and Y.Q. carried out cryo-EM data collection. Y.G. processed the cryo-EM data and built and refined the atomic model. Y.G., Y.Q. and Y.Z. analyzed the structure. B.H. and Q.B. conducted the molecular dynamic simulations. Y.G. wrote the original draft of the manuscript and prepared the figures. Y.Z., Y.G. and Y.Q. edited the manuscript.

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Correspondence to Yan Zhao.

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Extended data

Extended Data Fig. 1 Protein purification of human CHT1.

a. Size-exclusion chromatography of purified human CHT1 protein sample in NaCl/LMNG. Fraction within dashed lines were pooled for cryo-EM sample preparation. b. SDS-PAGE gel of purified human CHT1. Band smears because of heterogeneity caused by N-linked glycosylation. Purification of human CHT1 in the presence (c–d) and absence (e–f) of 40 mM choline using KCl buffer. c,e. Size-exclusion chromatography of purified human CHT1 protein sample (black) and reconstituted CHT1 in nanodiscs (red). Fraction within dashed lines were pooled for nanodisc reconstitution or cryo-EM sample preparation. d,f. SDS-PAGE gel of purified human CHT1 and MSP1D1E3. Band smears because of heterogeneity caused by N-linked glycosylation. The experiments were repeated independently for more than 3 times with identical results.

Source data

Extended Data Fig. 2 Cryo-EM data processing of dimeric CHT1HC-3/OF.

a. Data processing pipeline for the human dimeric CHT1 in the outward-facing state (CHT1HC-3/OF). A representative micrograph is shown here (Bar = 400 Å). A ‘seed-facilitated’ data processing strategy were used (see Method section for details). Particles were cleaned using multiple rounds of classifications. Monomeric CHT1 were processed separately (see Method section and Extended Data Fig. 3). The final map was reported at 3.6 Å according to GSFSC criterion. b. Angular distribution of the human dimeric CHT1 reconstruction. The height of each spike indicates the particle number at designated orientations. c. Sharpened map of dimeric CHT1, colored according to the estimated value of local resolution. d. Fourier shell correlations (FSC) curves of the dimeric CHT1 and its atomic model. The unmasked (blue) and masked (red) FSC curves was calculated between two independently refined half-maps before and after map sharpening. The model-vs-map FSC (black) was calculated between the full map and the atomic model. e. Representative cryo-EM density map. Critical residues are labeled. f. Different views of the model within the map.

Extended Data Fig. 3 Cryo-EM data processing of monomeric CHT1HC-3/OF.

a. Data processing pipeline for the human monomeric CHT1 in the outward-facing state (CHT1HC-3/OF). A representative micrograph is shown here (Bar = 400 Å). Particles were cleaned using multiple rounds of classifications. The final map was reported at 3.6 Å according to GSFSC criterion. b. Angular distribution of the monomeric CHT1 reconstruction. The height of each spike indicates the particle number at designated orientations. c. Sharpened map of monomeric CHT1, colored according to the estimated value of local resolution. d. Fourier shell correlations (FSC) curves of the monomeric CHT1 and its atomic model. The unmasked (blue) and masked (red) FSC curves was calculated between two independently refined half-maps before and after map sharpening. The model-vs-map FSC (black) was calculated between the full map and the atomic model. e. Representative cryo-EM density map. Critical residues are labeled. f. Different views of the model within the map.

Extended Data Fig. 4 Assembly of the dimeric CHT1HC-3/OF.

a. Cartoon representation of dimeric CHT1HC-3/OF in the side view (up) and the top-down view (down). The two protomers are colored pink and cyan, respectively. N-glycans and CHS are shown as gold sticks. LMNG are shown as green sticks. Dimeric interactions at the extracellular side were indicated using a dashed box and elaborated in (b). b. Zoomed-in view of the interaction at the dimer interface. CHS is shown as gold sticks. LMNG is shown as green sticks, overlaid with transparent grey surface. Residues involved in interactions with LMNG are shown as sticks and labeled. c. Interactions between the two protomers at the intracellular side of dimeric CHT1. Residues that may contribute to CHT1 dimerization were shown as sticks and labeled. d. Superimposition between the dimeric (pink and cyan) and monomeric (grey) CHT1 in the outward-facing state.

Extended Data Fig. 5 Surface expression of CHT1 WT and mutants.

a. Left, β-actin protein was only detected in the cell lysate rather than the surface protein elutes, indicating that the intracellular proteins were not biotinylated. The Na+/K+ ATPase protein was detected in the elution sample from biotinylated cells, indicating that the plasma membrane proteins can be effectively biotinylated and purified using streptavidin beads. FLAG-tagged CHT1 was observed in the lysate of cells expressing CHT1 WT and in the elution from streptavidin beads, but not in cells without CHT1 expression or in unbiotinylated cells, confirming that the biotinylated CHT1 can be detected using the anti-FLAG antibody. Right, immunoblots of biotinylated CHT1 WT and mutants on cell surface. Biotinylated Na+/K+ ATPase on the cell surface was used as the loading control. b. Densitometric quantification of surface expression of CHT1 WT and mutants (glycosylated and non-glycosylated), normalized using density values of Na+/K+ ATPase within a single batch of experiment, and mean density values of all bands (CHT1 WT and mutants) across independent batches of experiments. Each triangular symbol indicates a single data point. Data are represented as mean ± S.E.M. n = 4 biologically independent experiments.

Source data

Extended Data Fig. 6 Cryo-EM data processing of CHT1apo/IF.

a. Data processing pipeline for human CHT1 in the inward-facing apo state (CHT1apo/IF). A representative micrograph is shown here (Bar = 400 Å). Particles were cleaned using multiple rounds of classifications. The final map was reported at 3.7 Å according to GSFSC criterion. b. Angular distribution of the CHT1 reconstruction in the inward-facing apo state. The height of each spike indicates the particle number at designated orientations. c. Sharpened map of CHT1 in the inward-facing apo state, colored according to the estimated value of local resolution. d. Fourier shell correlations (FSC) curves of the inward-facing apo CHT1 and its atomic model. The unmasked (blue) and masked (red) FSC curves was calculated between two independently refined half-maps before and after map sharpening. The model-vs-map FSC (black) was calculated between the full map and the atomic model. e. Representative cryo-EM density map. Critical residues are labeled. f. Different views of the model within the map.

Extended Data Fig. 7 Cryo-EM data processing of CHT1choline/occ.

a. Data processing pipeline for human CHT1 in the choline-bound inward-facing occluded state (CHT1choline/occ). A representative micrograph is shown here (Bar = 400 Å). Particles were cleaned using multiple rounds of classifications. The final map was reported at 3.7 Å according to GSFSC criterion. b. Angular distribution of the CHT1 reconstruction in the inward-facing occluded state. The height of each spike indicates the particle number at designated orientations. c. Sharpened map of CHT1 in the inward-facing occluded state, colored according to the estimated value of local resolution. d. Fourier shell correlations (FSC) curves of the inward-facing occluded CHT1 and its atomic model. The unmasked (blue) and masked (red) FSC curves was calculated between two independently refined half-maps before and after map sharpening. The model-vs-map FSC (black) was calculated between the full map and the atomic model. e. Representative cryo-EM density map. Critical residues are labeled. f. Different views of the model within the map.

Extended Data Fig. 8 Structural comparison between CHT1apo/IF and CHT1choline/occ and choline binding site.

a–b. Superimposition between the CHT1apo/IF (blue) and CHT1choline/occ (red). c–d. Binding pocket of choline in the inward-facing CHT1 structures, overlaid with their corresponding density shown in blue mesh. Choline and critical residues that may contribute to choline recognition are labeled. e. Backbone R.M.S.D. of CHT1 (excluding choline and high flexibility loop residue 27–49 and 501–517) from the initial structure and the heavy atom R.M.S.D. of choline from its initial position are plotted over time. f. The representative structure obtained from the clustering analysis involving three 120 ns independent trajectories. Only one cluster was observed considering choline and the pocket (i. e. protein residues within 5 Å of the choline) and using 1.5 Å as the R.M.S.D. cut-off. The hydrogen bonds between the hydroxyl group of choline and residues W62, W406 are indicated with dashed lines. g. Statistical analysis of hydrogen bonds between choline and the residues in the choline binding pocket for Trajectory 1–3 and the concatenated trajectory. Hydrogen bonds were detected with a polarity heavy atom donor-acceptor distance of 3.5 Å and an angle cut-off of 40°.

Source data

Extended Data Fig. 9 Structural analysis of CHT1 and SMCT1.

a. Superimposition between CHT1apo/IF (blue) and the inward-facing SMCT1 (grey). b. ThermoFluor curves of the IH1/(GS)3 substitution (CHT1IH1/GS) mutant. CHT1IH1/GS respond to choline but failed to exhibit sensitivity to HC-3 in the NaCl or KCl buffers.

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Extended Data Fig. 10 Structure analysis of the sodium binding sites.

a–b. Structural comparison between NIS (gray) and CHT1choline/occ (red). Na1 and the non-canonical NIS Na2 sites of NIS were labeled. The sodium ions and the iodide ion were shown as spheres and labeled. Residues interacting with the ions are shown as sticks and labeled in (b). c–d. Structural comparison between the outward-facing SiaT (grey) and CHT1HC-3/OF (green). Sodium ions at the Na2 (c) and Na3 (d) sites of SiaT were shown as spheres and labeled. Critical residues within the sodium binding sites are shown as sticks or spheres (G64 Cα atom) and labeled. Residues constituting the sodium binding sites via backbone carbonyl were labeled within square brackets. e. Sequence alignment of canonical Na2 and canonical Na3 sites between CHT1 homologues. f. Structures of CHT1HC-3/OF (green) and CHT1choline/occ (red), overlaid with transparent grey surfaces. Canonical sodium binding sites Na2 and Na3 sites (SiaT nomination) are labeled. Residues constituting the binding sites were shown as sticks. The Na3 site are occluded in the outward-facing open CHT1HC-3/OF (left) and exposed to the cytosol in the inward-facing occluded CHT1choline/occ (right).

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Qiu, Y., Gao, Y., Huang, B. et al. Transport mechanism of presynaptic high-affinity choline uptake by CHT1. Nat Struct Mol Biol 31, 701–709 (2024). https://doi.org/10.1038/s41594-024-01259-w

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