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Structure and inhibition mechanism of the human citrate transporter NaCT

Nature volume 591, pages 157–161 (2021)Cite this article

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Abstract

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1,2,3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor—which binds to the same site as citrate—arrests the transport cycle of NaCT. The NaCT–inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6,7,8.

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Fig. 1: Biochemical characterization and structure determination of human NaCT.
Fig. 2: Na+- and substrate-binding sites in human NaCT and mapping of SLC13A5 mutations that cause epilepsy.
Fig. 3: PF2-binding site in human NaCT.
Fig. 4: Inhibition mechanism of PF2.

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Data availability

Electron microscopy densities and protein models have been deposited in the Electron Microscopy Data Bank and Protein Data Bank for the NaCT–citrate (EMD-22457, 7JSK) and NaCT–PF2 (EMD-22456, 7JSJ) complexes. All other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by the NIH (R01NS108151 and R01GM121994), the G. Harold and Leila Y. Mathers Foundation, the TESS Research Foundation, the American Epilepsy Society and Pfizer. D.B.S. was supported by the American Cancer Society Postdoctoral Fellowship (129844-PF-17-135-01-TBE) and Department of Defense Horizon Award (W81XWH-16-1-0153). J.K.H. and J.A.M. were supported by the NINDS Intramural Program. We thank the following colleagues for reagents, technical assistance and discussions: Y. Cheng, N. Coudray, K. Huard, T. Kawate, J. Li, R. Mancusso, J. Marden, F. Ono and J. Zhao; the staff at the following facilities for assistance in grid screening and data collection: K. Maruthi at the Simons Electron Microscopy Center at the New York Structural Biology Center, H. Scott at the Pacific Northwest Center for Cryo-EM and K. Dancel from the NYU Microscopy Core. Electron microscopy data processing used computing resources at the HPC Facility of NYULMC, and we were assisted by A. Siavosh-Haghighi and M. Costantino.

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Author notes

  1. Nathan K. Karpowich

    Present address: Janssen Pharmaceuticals, Spring House, PA, USA

Authors and Affiliations

  1. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    David B. Sauer, Jinmei Song, Nathan K. Karpowich & Da-Neng Wang

  2. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    David B. Sauer, Jinmei Song, Nathan K. Karpowich, William J. Rice & Da-Neng Wang

  3. Cryo-Electron Microscopy Core, New York University School of Medicine, New York, NY, USA

    Bing Wang & William J. Rice

  4. Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

    Jacob K. Hilton & Joseph A. Mindell

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Contributions

J.S. expressed and purified the protein. J.S., N.K.K. and J.K.H. conducted biochemical studies. D.B.S. froze grids. D.B.S., B.W. and W.J.R. collected and processed the cryo-EM images. D.B.S. built the atomic models. D.B.S and D.-N.W. analysed the structures. D.B.S., J.K.H., J.A.M., W.J.R. and D.-N.W. wrote the manuscript. All authors participated in the discussion and manuscript editing. D.-N.W. supervised the research.

Corresponding authors

Correspondence to Joseph A. Mindell, William J. Rice or Da-Neng Wang.

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The authors declare no competing interests.

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Peer review information Nature thanks Raimund Dutzler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Kinetic cycle of NaCT and molecular structures of its substrates and inhibitors.

a, b, Kinetic cycle (a) and schematic model (b) of the SLC13 transport cycle. Co, outward-facing conformation; Ci, inward-facing conformation; S, substrate. The number of cotransported Na+ for different SLC13 transporters varies between 3 and 4, but only two are shown here. All available biochemical evidence indicates that sodium ions bind before and release after the substrate. c, Molecular structures of the NaCT substrate, citrate and various inhibitors. d, IC50 of inhibitors for NaCT and the dicarboxylate transporters NaDC1 and NaDC323,24,25. Whereas PF2 is highly selective towards NaCT, PF4 and PF4a inhibit all three human di-/tricarboxylate transporters.

Extended Data Fig. 2 Purification and functional characterization of human NaCT.

a, Michaelis–Menten plot showing the citrate dependence of Na+-driven radioactive citrate uptake into HEK293 cells that expressed eGFP–NaCT. HEK293 cells transfected with an eGFP vector were used as a control. All data points include six biological replicates from two independent experiments, with error bars indicating the s.d. Inset, representative confocal image of HEK293 cells transfected with the eGFP–NaCT construct, from four biological replicates. Scale bar, 10 μm. b, Analytical fluorescence SEC of detergent-solubilized cell lysate of Hi5 cells overexpressing an eGFP–NaCT construct. Peak height represents the protein concentration, whereas the peak sharpness indicates the protein homogeneity. The cell lysate was solubilized in DDM detergent, incubated with various compounds at 37 °C, and loaded onto an analytical SEC column on HPLC. c, Preparative SEC of NaCT following Ni2+-NTA affinity purification. d, Representative SDS–PAGE of purified NaCT, from twenty biological replicates. e, NaCT binding to citrate in detergent solution as measured by tryptophan fluorescence quenching. All points include three biological replicates, with error bars indicating the s.d. The Kd was found to be 148 ± 28 μM. f, Molecular mass measurements of DDM-purified NaCT using multi-angle light scattering. The measured mass of 125 ± 2 kDa agrees with the molecular weight of a dimeric NaCT of 126.124 kDa calculated from the protein sequence.

Source data

Extended Data Fig. 3 Characterization of the NaCT–citrate cryo-EM specimens and flow chart of image processing.

a, Violin plot showing the distribution of ice thickness in electron micrographs from specimens tilted at 0°, 20°, 40° and 50°. The plot widths correspond to ice thickness distribution. Theoretically, the ice thickness at 20°, 40° and 50° tilts would increase from 0° by 6%, 30% and 56%, respectively. The actual number of electron micrographs with ultra-thin ice (5–20 nm) decreased significantly with the tilt angle. b, Violin plot showing the distribution of the average horizontal particle displacements from the first five frames of each electron micrograph. The beam-induced particle displacements increased with the tilt angle. c, Violin plot showing the distribution of micrograph CTF fit resolution of the micrographs. The image quality markedly deteriorated for those recorded from 50° tilted specimens. d, Flow chart of image processing of the NaCT–citrate images. Only images collected from specimens tilted at 0°, 20° and 40° were included in the processing and the generation of the finals maps.

Extended Data Fig. 4 Cryo-EM data collection from 0°, 20° and 40° tilted specimens and image processing of the NaCT–citrate complex.

a, Orientation distribution of particles from a NaCT–citrate complex reconstruction using only particles from 0° tilt micrographs. At 0° sample tilt most of the particles are top views (viewed along the membrane normal). Side views (viewed from within the membrane plane) are relatively rare. The number of side views and top views differ by three orders of magnitude, indicating a considerable degree of preferred orientation. b, c, Orientation distribution of particles from a NaCT–citrate complex reconstruction using particles from 40° specimen tilt (b) and all micrographs at 0°, 20° and 40° specimen tilts (c). With tilting, the orientation distribution of particles becomes much more isotropic, alleviating the preferred orientation problem. d, e, The 30 most populous classes from two-dimensional classification of particles from the 0° (d) and 40° (e) tilted specimens. The 0° classes are dominated by top views, with few side and oblique views. By contrast, the 40° micrographs include clear side- and oblique-view classes.

Extended Data Fig. 5 Structure determination of NaCT–citrate complex.

a, Cryo-EM map FSC curve of the NaCT–citrate complex reconstruction using all micrographs. Arrows indicate the nominal map resolution of 3.04 Å, based on a threshold of FSC = 0.143. b, Directional FSC curves of the NaCT–citrate complex reconstruction. Each purple trace is an individual FSC calculated from a conical wedge of the overall spherical shell, sampled on a 500-point Fibonacci spherical grid. The global FSC curve (the yellow trace), as calculated by averaging all directional FSC curves, also indicates a resolution of 3.04 Å. c, Mask used for refinement using cryoSPARC. d, Local resolution of the map. e, Example cryo-EM densities showing the quality of the chain tracing of the NaCT–citrate model. All of the key helices that are involved in citrate and sodium ion binding are shown. The density for peripheral helix TM1 is poorly resolved, with the helix loosely attached to the rest of the protein. f, Model of NaCT dimer. The scaffold domain and the transport domain in each protomer is coloured green and pink, respectively. g, Model of the NaCT protomer as viewed from the cytosol. C1 symmetry was used for the image reconstruction and model refinement. The two protomers are identical, with a root mean square deviation of 0.002 Å.

Extended Data Fig. 6 Features of the NaCT–citrate structure.

a, Cryo-EM density map around the citrate-binding sites. All of the densities are shown at the same contour levels. The density for citrate is coloured red. b, Electrostatic surface of the citrate-binding site. The sodium ions at Na1 and Na2 were included in the calculations. c, Overlay of the NaCT–citrate and VcIndy–succinate (PDB: 5UL7) structures, along with their respective substrates, shown in green and grey, respectively. d, Locations of the SLC13A5-epilepsy missense mutations within the NaCT structure as viewed from the cytosol. e, Sequence alignments of the first SNT motif (left), L5ab–TM5b (centre) and second SNT motif (right) of SLC13 family proteins and bacterial homologues. The second SNT motif in NaCT has a sequence of Ser-Asn-Val. f, Interaction of Lys107 and Arg108 on H4c with other residues on H6b and TM7. g, Aromatic clusters near TM6.

Extended Data Fig. 7 Cryo-EM data collection from tilted specimens and reconstruction FSC curve of the NaCT–PF2 complex.

a, Orientation distribution of particles from a NaCT–PF2 complex reconstruction using only particles from images of 0° tilt specimens. At 0° sample tilt most of the particles are top views, whereas side views are relatively rare. The number of side views and top views differ by up to three orders of magnitude, indicating a considerable degree of preferred orientation. b, c, Orientation distribution of particles from a NaCT–PF2 complex reconstruction using particles from 40° tilt (b) and all micrographs collected at 0°, 20° and 40° tilts (c). With tilting, the particle views become much more isotropic, alleviating the preferred orientation problem. d, Cryo-EM map FSC curve of the NaCT–PF2 complex reconstruction using all micrographs. e, Cryo-EM map of the NaCT–PF2 complex with a resolution of 3.12 Å. f, Local resolution of the map. g, Example cryo-EM densities showing the quality of the chain tracing of the NaCT–PF2 model. All of the key helices that are involved in PF2 and sodium ion binding are shown.

Extended Data Fig. 8 Map and structural model of the NaCT–PF2 complex.

a, b, Structure of the NaCT–PF2 complex as viewed from the membrane plane (a) and the cytosol (b). c, d, Cryo-EM density map around the PF2-binding sites. All of the densities are shown at the same contour levels. The density for PF2 is coloured red. e, PF2-binding site as viewed from within the transport domain. f, Packing of the scaffold domain side chains around PF2. The scaffold and transport domains are coloured green and pink, respectively. Residues Leu56, Ala57, Gly409 and Ile410 are shown as spheres. g, Overlay of the NaCT–citrate and NaCT–PF2 structures in green and blue, respectively. The loops enclosing Na1 and Na2 sodium-binding sites move by around 1 Å, more tightly enclosing both sites in the NaCT–PF2 complex. h, Na+-driven citrate uptake into HEK293 cells transfected with various eGFP-tagged NaCT mutants. Each data point includes three biological replicates, with error bars indicating the s.d. NaCT(G409Q) and NaCT(I410V) mutants retained wild-type level activity and were used to measure inhibition by PF2 in Fig. 4b.

Extended Data Table 1 Cryo-EM data collection and structure determination of NaCT
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Extended Data Table 2 Classification of SLC13A5-epilepsy mutations
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Sauer, D.B., Song, J., Wang, B. et al. Structure and inhibition mechanism of the human citrate transporter NaCT. Nature 591, 157–161 (2021). https://doi.org/10.1038/s41586-021-03230-x

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