Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5

Abstract

Cancer cells undergo a shift in metabolism where they become reliant on nutrients such as the amino-acid glutamine. Glutamine enters the cell via the alanine/serine/cysteine transporter 2 (ASCT2) that is upregulated in several cancers to maintain an increased supply of this nutrient and are therefore an attractive target in cancer therapeutic development. ASCT2 belongs to the glutamate transporter (SLC1A) family but is the only transporter in this family able to transport glutamine. The structural basis for glutamine selectivity of ASCT2 is unknown. Here, we identify two amino-acid residues in the substrate-binding site that are responsible for conferring glutamine selectivity. We introduce corresponding mutations into a prokaryotic homologue of ASCT2 and solve four crystal structures, which reveal the structural basis for neutral amino acid and inhibitor binding in this family. This structural model of ASCT2 may provide a basis for future development of selective ASCT2 inhibitors to treat glutamine-dependent cancers.

Structural basis for the transport mechanism of the human glutamine transporter SLC1A5 (ASCT2)

“…In the outward-facing state, there are two observed conformational populations. One is represented by the ECL2a encircling the transport domain (PDB: 1XFH, 2NWX, 2NWW, 2NWL, 4IZM, 6BMI, 6BAU, 6BAVand 6BAT) as observed in our SLC1A5 cryo-EM structures (6MP6 and 6MPB) (7,8,22,23). The other population is represented by the ECL2a burying ECL4 and ECL5 (PDB: 4OYE, 5CFY, 4OYF, 5E9S, 5DWY and 4KY0) (13,14,18).…”

“…Besides the difference in the pocket size and shape, Arg in place of the Cys will lead to steric clashes with the Gln substrate in SLC1A5. The critical role of the Cys467 in SLC1A5 for substrate specificity was further supported by an equivalent mutation of threonine in SLC1A4 (Thr459) to a cysteine, resulting in alteration to L-Gln binding (22) and inhibition by Cys modifying reagents in proteoliposome assays (27). Ala390, a conserved residue in neutral amino acid transporters, SLC1A4 and SLC1A5, does not form any contact with the Gln substrate and could tolerate small neutral amino acid substrates (Fig.…”

“…S12, Table S2) (7-9, 11, 14-16, 18, 19, 22-24). In inhibitor-bound structures, the HP2 loops are largely either open or disordered (8,15,22). For structures in the absence of small molecule ligands, including sodium-bound and unbound states the HP1-2 tip distance varies (13,14,18).…”

Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism

“…Glutamine serves as a crucial metabolic substrate in cancer cell and participates in tumour development and proliferation. Alanine‐serine‐cysteine transporter 2 (ASCT2), the membrane‐associated protein, is a broad specificity bidirectional transport of glutamine . As the mediator of glutamine uptake and metabolism, ASCT2 is involved in the aetiology of multiple cancers, such as breast cancer, prostate cancer and melanoma.…”

“…Alanine-serine-cysteine transporter 2 (ASCT2), the membrane-associated protein, is a broad specificity bidirectional transport of glutamine. 2 As the mediator of glutamine uptake and metabolism, ASCT2 is involved in the aetiology of multiple cancers, such as breast cancer, prostate cancer and melanoma. It was reported that the inhibition of ASCT2 repressed reduced cancer cell growth and proliferation in LS174T cell line.…”