Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. This domain detachment may be required for substrate binding and release on the intracellular side of the membrane.
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We thank C. Indivieri (Università della Calabria, Italy) for kindly providing the P. pastoris expression strain, R.C. Prins for her help in preparation of cholesterol-containing proteoliposomes, M. Punter for help in setting up the image-processing cluster and B. Poolman for critical reading and discussion of the manuscript. This research was supported by NWO Vidi grant 723.014.002 to A.G.; NWO Veni grant 722.017.001 and Marie Skłodowska–Curie Individual Fellowship 749732 to C.P.; and NWO Vici grant 865.11.001 and European Research Council Starting Grant 282083 to D.J.S. C.G. thanks the SLAC National Accelerator Laboratory for financial support as part of the Panofsky fellowship program.
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Cryo-EM reconstruction of ASCT2.
a,b, Representative cryo-EM image (a) and 2D-class averages (b) of vitrified ASCT2. c, Angular distribution plot of particles included in the final C3-symmetrized 3D reconstruction. The number of particles with the respective orientations is represented by length and colour of the cylinders (long and red: high number of particles; short and blue: low number of particles). d, Image processing work flow. e, Analysis of conformational heterogeneity in the dataset. 3D classification without any symmetry imposed where performed on the indicated particle dataset: after several rounds of 2D classification (I, on 628,015 particles) and after several rounds of 3D classification (II, on 307,619 particles). Particles obtained before particle polishing, which rendered a map of 3.9Å resolution were used to perform symmetry expansion followed by particle subtraction on the individual protomers, yielding a dataset of 552,240 particles (III). Resulting classes of the 3D classification without image alignment are shown. In all cases only the inward-facing state of ASCT2 was identified. f, Final reconstruction map coloured by local resolution as estimated by Relion. g, FSC plot of the final refined unmasked (grey) and masked (blue) map. The resolution at which the curve drops below the 0.143 threshold is indicated. A thumbnail of the mask used for FSC calculation overlaid on the atomic model is shown in the upper right corner. h, FSC curves of the refined model versus the map of ASCT2 for cross-validation. The purple shows the FSC curves for the refined model compared to the full masked dataset (FSCsum); light grey, FSC curve for the refined model compared to the masked half-map 1 (FSCwork, used during validation refinement); dark grey, refined model compared to the masked half- map 2 (FSCfree, not used during validation refinement). Dashed lines, FSC threshold used for FSCsum of 0.5 and for FSCfree/work of 0.143.
Supplementary Figure 2 Cryo-EM density.
Sections of the cryo-EM density of the ASCT2 map superimposed on the respective refined models. Models are shown as sticks and structural elements are labelled. Transmembrane helices of the transport domain are coloured in blue, of the scaffold domain in yellow and the loop between TM4b and TM4c is shown in red. Densities sharpened with a b-factor of −225 Å2 were plotted at 5σ, except for the loop between TM4b-TM4c, which was contoured at 3σ.
Supplementary Figure 3 Superposition and orientation of domains in ASCT2 and EAAT1.
Superposition of transport (a) and scaffold (b) domains of ASCT2 (bright blue and bright yellow) with EAAT1 (light blue and light orange, PDB 5LLU), respectively. Rmsds are ~ 1Å. c,d, The scaffold domains of ASCT2 (yellow ribbon) and EAAT1 (orange ribbon, PDB 5LLU) were aligned structurally, revealing large differences in the position of the transport domains (dark and light blue surfaces, respectively) c, The movement form the outward (as observed in EAAT1) to the inward (as seen in ASCT2) state is indicated with an arrow. d, A pair of equivalent residues (Glu444 of ASCT2 and T456 of EAAT1) is highlighted in red to emphasize the movement of ~ 25 Å.
Supplementary Figure 4 Visualization of the domain interface and putative lipid density.
A single protomer of ASCT2 (a), EAAT1 (PDB 5LLU) (b), GltPh in the locked state (PDB 4X2S, chain A) (c) and GltPh in the unlocked state (PDB 4X2S, chain C) (d) viewed from the membrane plane. The scaffold domain is depicted as yellow ribbon and the transport domain in grey surface representation) with the interacting parts in black. The surface area of the interfaces are extensive in EAAT1 and locked GltPh. The transport domains in ACST2 and unlocked GltPh are more detached from the scaffold domain, but the shapes of their interaction surfaces are different, with most of the interacting residues in ASCT2 located in a narrow strip on the extracellular side. e, Patches of densities (green mesh at 4σ) observed between scaffold (yellow) and transport domain (blue) can accommodate cholesteryl hemisuccinate molecules shown as pink sticks, but the identities of the molecules could not be assigned unambiguously.
Supplementary Figure 5 Pseudosymmetrical relation between the transport domains of EAAT1 and ASCT2.
Transport domain of the outward-facing structure of EAAT1 (PDB 5LLU, pale colors, panel a, d), and the inward-facing structure of ASCT2 (bright colors, panel c, f) were structurally aligned on their scaffold domains (not visible) and are depicted from two perpendicular viewpoints in the plane of the membrane. b,e: The pseudo-symmetrical organization observed between transport domains in both structures becomes apparent when they are overlaid. TM3 (bright blue) is pseudo-symmetrical to TM6 (pale blue), TM6 (bright pink) to TM3 (pale pink) and HP1 (bright yellow and orange) to HP2 (pale yellow and pale orange). In the top row the pseudo two-fold axis is indicated as a dashed arrow. In the bottom row the two-fold axis is indicated as a black dot and points towards the reader.
Supplementary Text and Figures
Supplementary Figures 1–5
Sequence alignment. Sequence alignment of human ASCT2 with all other human SLC1 family members and archaeal homologues GltPh and GltTk. Transmembrane segments are indicated with numbered cylinders and colored yellow and blue for scaffold and transport domains, respectively. The protruding loop forming the ‘antenna’ is shown with the red dashed box. The sequence conservation is shown with different shades of violet. The residues forming a putative salt bridge between the domains in ASCT2 and the binding-site residues are indicated by grey squares and triangles, respectively. The transport domain of ASCT2 shares 50% and 34% identical residues with those of EAAT1 and GltPh, respectively. The scaffold domains 42% and 27%, respectively.
Supplementary Video 1
ASCT2 structure. The cryo-EM density (sharpened with a B-factor of -225 Å2) of the human inward-facing ASCT2 amino acid exchanger, is shown with the modelled structure superimposed. For clarity only one protomer of the trimer is shown, the transport domain is coloured in blue, the scaffold domain in yellow and the extracellular loop between TM4b and TM4c is shown in red. The view is from within the membrane.
Supplementary Video 2
Putative conformational changes of the transport domain in human SLC1. Morph between the inward-facing human ASCT2 structure and the outward-facing human EAAT1 (PDB-ID: 5llm) structure. The structures were superimposed on their scaffold domains. For clarity only one protomer of the homotrimer is shown. The scaffold domain is coloured in yellow and the transport domain in blue with the hairpins HP1 depicted in light purple and HP2 in purple. The hairpins of the inward-facing unlocked GltPh (PDB-ID: 4X2S, chain C) structure are shown in green. The membrane boundaries are indicated by grey lines. Note the accessibility of HP2 to the cytoplasm.
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Garaeva, A.A., Oostergetel, G.T., Gati, C. et al. Cryo-EM structure of the human neutral amino acid transporter ASCT2. Nat Struct Mol Biol 25, 515–521 (2018). https://doi.org/10.1038/s41594-018-0076-y
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