Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

Abstract

The L-type amino acid transporter 1 (LAT1 or SLC7A5) transports large neutral amino acids across the membrane and is crucial for brain drug delivery and tumor growth. LAT1 forms a disulfide-linked heterodimer with CD98 heavy chain (CD98hc, 4F2hc or SLC3A2), but the mechanism of assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1–CD98hc heterodimer at 3.3-Å resolution. LAT1 features a canonical Leu T-fold and exhibits an unusual loop structure on transmembrane helix 6, creating an extended cavity that might accommodate bulky amino acids and drugs. CD98hc engages with LAT1 through the extracellular, transmembrane and putative cholesterol-mediated interactions. We also show that two anti-CD98 antibodies recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities. These results reveal the principles of glycoprotein-solute carrier assembly and provide templates for improving preclinical drugs and antibodies targeting LAT1 or CD98hc.

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Fig. 1: Architecture of LAT1–CD98hc.
Fig. 2: Structure of LAT1.
Fig. 3: Structural basis for broad substrate specificity.
Fig. 4: Anti-CD98 antibodies and epitopes.
Fig. 5: Model of amino acid antiport by LAT1–CD98hc.

Data availability

The atomic coordinates of LAT1–CD98hc–MEM-108 Fab and CD98hc-ED–HBJ127 Fab–MEM-108 Fab have been deposited in the Protein Data Bank under accession numbers 6JMQ and 6JMR. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-9849 and EMD-9850. The raw micrographs used for this study are available at the Electron Microscopy Public Image Archive under accession codes EMPIAR-10264 and EMPIAR-10265. Source data for Fig. 3d,e and Supplementary Figs. 1d–g and 5a–c are available online. All other data are available from the corresponding author upon request.

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Acknowledgements

We thank R. Danev and M. Kikkawa for setting up the cryo-EM infrastructure; K. Ogomori and M. Miyazaki for technical assistance; G. Kasuya, M. Fukuda and R. Taniguchi for discussions; and W. Kühlbrandt for comments on the manuscript. Y.L. was supported by the Toyobo Biotechnology Foundation Fellowship. This work was supported in part by MEXT/JSPS KAKENHI under grant numbers JP16J07405, to Y.L., and JP16H06294, to O.N.; by AMED under grant numbers JP18am0101082, to M.S., JP18gm0810010, to S.N., JP18cm0106131, to Y.K., and JP18am0101115, to O.N.

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Authors

Contributions

Y.L. and O.N. conceived the study. Y.L. performed expression screening, purified LAT1–CD98hc and Fab, prepared the cryo-EM samples and determined the structure. Y.L., T.K., T.Ni., R.I., T.Y. and M.S. collected the cryo-EM data. P.W. and S.N. performed the transport assays using proteoliposomes. C.J., L.Q., R.O., S.O. and Y.K. performed the transport assays using X. laevis oocytes. T.Na assisted with high-resolution cryo-EM data processing with RELION. K.O. purified the proteins for biochemical assays. H.E. provided LAT1 inhibitors and assisted with data interpretation. Y.L. and O.N. wrote the manuscript with contribution from all authors. O.N. supervised the project.

Corresponding author

Correspondence to Osamu Nureki.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Purification and biochemical characterization of LAT1-CD98hc.

a, Size-exclusion chromatography profile of LAT1-CD98hc. The fractions used for structural and functional analyses are indicated by a black bar. b, SDS-PAGE analyses of LAT1-CD98hc with or without 20 mM β-ME. c, Chemical structures of LAT1 inhibitors. d, Uptake of l-[3H]Tyr (50 μM, 10 Ci/mol) by purified LAT1-CD98hc reconstituted in liposomes (cyan) or by control liposomes (white). Proteoliposomes preloaded with 4 mM l-Gln (rectangle) exhibited an initial overshoot of uptake (~1–10 min) as compared to those without preloading (circle), indicating the antiport reaction. The proteoliposomes without preloading also slowly accumulated l-[3H]Tyr, suggesting that LAT1 could adopt facilitative transport mode, in addition to the antiport mode. Values are mean ± SEM. n = 3 technical replicates. e, Substrate specificity of LAT1-CD98hc. Proteoliposomes were incubated with various radiolabeled amino acids and the uptake was measured at 10 min. Control liposomes were used for background subtraction. Although we were not able to measure effective uptake of radiolabeled l-Leu and l-Kyn here due to the high background (Source Data), the competitive inhibition assay confirmed the strong inhibition by l-Leu (f), supporting its transport. Values are mean ± SEM. n = 3 technical replicates. f, Inhibition assay. Proteoliposomes were incubated with l-[3H]Tyr (10 μM) in the presence of 30 mM competitive inhibitors in the external solution. Values are mean ± SEM. n = 3 technical replicates. g, Inhibition assay with pre-incubation of 30 μM SKN-102, SKN-103 and JPH203, or 30 mM BCH. h, Tryptophan-fluorescence size exclusion chromatography of LAT1-CD98hc-Fab complexes. I, Representative 2D class averages of LAT1-CD98hc with or without Fab, recorded on a 200 kV JEM2010F microscope equipped with a CCD camera. Fab molecules are highlighted by yellow asterisks. Note that the box sizes are different between the images with or without Fab.

Supplementary Figure 2 Atomic model of LAT1-CD98hc in the cryo-EM density map.

a, Cryo-EM density maps and atomic models are shown for selected regions. TM1–TM12, IL1 and EL2–EL4 of LAT1 (cyan), TM1’ and the linker of CD98hc (green). The disulfide bond, four N-glycans (orange) and five lipids (yellow). b, Density map of LAT1 in different views. Sterol densities are colored yellow. c, Stereo view of TM1a-TM1b and TM6a-TM6b. The EM map shows no prominent density in the putative substrate-binding site, indicating the apo state. The similar view is shown in Fig. 2c.

Supplementary Figure 3 LAT1-CD98hc interaction.

a, Correlation with previous cross-linking data on LAT2-CD98hc37. Upper, cross-linked pairs are shown on the structure as lines. Dotted lines are for a 60% cross-linked pair. Lower, cross-linked pairs in CD98hc and LAT2 are summarized in a table, along with the corresponding residues in LAT1 and the measured Cα-Cα distances. N/A, not applicable because G220 is disordered, and for visualization I219 is used instead. b, Surface electric potential calculation of LAT1 and CD98hc.

Supplementary Figure 4 Topology of LAT1 and its inward-open state.

a, Side view of LAT1. Helices are colored from blue to red. b, Topology diagram of LAT1. The two large triangles indicate the ‘5+5’ inverted repeats. c, Extracellular and cytoplasmic gates. d, Stereo view of the extracellular gate. e, Stereo view of the cytoplasmic gate. f, Comparison of TM1-TM6-TM10 between LAT1, LAT2, ApcT and AdiC. Unique configuration of the TM6a-TM6b loop underlies the specificity of LAT1. g, Sequence comparison of TM1 and TM6 in LAT1, ApcT and AdiC. The position of Gly255 is indicated by an red arrow. Blue boxes indicate the loop between the two discontinuous helices.

Supplementary Figure 5 Transport assays using X. laevis oocytes.

a, Uptake of l-[14C]Leu by X. laevis oocytes co-expressing CD98hc and LAT1 variants measured at four different substrate concentrations. The data used for Fig. 3d are marked by gray dotted rectangles. The specific radioactivities of l-[14C]Leu are 3.3, 3.3, 2.2 and 0.66 Ci/mol for 50, 100, 300 and 1,000 μM, respectively. Values are mean ± SEM. n = 5–9 technical replicates. b, Uptake of l-[14C]Ala. The specific radioactivities of l-[14C]Ala are 5.6, 5.6, 1.9 and 1.1 Ci/mol for 50, 100, 300 and 1,000 μM Ala uptake, respectively. Values are mean ± SEM. n = 4–9 technical replicates. c, Uptake of various radiolabeled amino acids by X. laevis oocytes co-expressing CD98hc and LAT1 (wild type or G255A) measured at two different substrate concentrations. The data from the 10 µM substrate concentrations are used for Fig. 3e. Values are mean ± SEM. n = 6–9 technical replicates. The specific radioactivities used for 100 µM are 2.2, 4.8, 4.5, 3.3, 20 and 1.1 Ci/mol for l-[14C]Trp, l-[14C]Tyr, l-[14C]Phe, l-[14C]Leu, [3H]Val and l-[14C]Ala, respectively. d, Western blotting of CD98hc and LAT1 variants expressed in X. laevis oocytes. Isolated oocyte membranes were fractionated by SDS-PAGE in the absence of DTT, and blotted with anti-LAT1 or anti-CD98hc antibodies. The results confirmed the heterodimer formation for all variants. The data are representative results from two independent experiments. e, Immunofluorescence of X. laevis oocytes, detected by an anti-LAT1 antibody. The results confirmed the plasma membrane localization for all variants.

Supplementary Figure 6 Full-length CD98hc and lipid interactions.

a, Crystal structure of CD98hc-ED (PDB: 2DH3). It superimposes with the present structure with a root mean square deviation (r.m.s.d.) of 1.31 Å for 414 Cα atoms. b, Cryo-EM structure of CD98hc in the LAT1-CD98hc heterodimer. c, Close-up view of the linker. Atom-atom distances are labeled in gray. Linker residues are labeled in red. d, Close-up view of the sterol binding sites. Densities are contoured at 11 σ.

Supplementary Figure 7 Analysis of SLC3-SLC7 interface.

a, The interaction between CD98hc and LAT1. b, TM1'-TM4 interface. c, Linker-Cβ2-Cβ3-Cβ8-EL2 interface. d, Aα8-EL4a and Aα1-Aα2-EL3 interface. e, Sequence alignment of EL2 and TM4. The conserved 9-amino acid stretch of EL2 is enclosed by a blue box. f, Sequence alignment of EL3. g, Sequence alignment of EL4a-EL4b. h, Sequence alignment of CD98hc and rBAT.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Notes 1 and 2

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Supplementary Video 1

Cryo-EM density map of LAT1 and CD98hc. The 360°-view of the density map and the atomic model are shown for the LAT1 and CD98hc TMD.

Source Data Fig. 3d,e, Supplementary Fig. 1d–g, Supplementary Fig. 5a–d

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Lee, Y., Wiriyasermkul, P., Jin, C. et al. Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc. Nat Struct Mol Biol 26, 510–517 (2019). https://doi.org/10.1038/s41594-019-0237-7

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