System x_c⁻ is an amino acid antiporter that imports L‐cystine into cells and exports intracellular L‐glutamate, at a 1:1 ratio. As L‐cystine is an essential precursor for glutathione synthesis, system x_c⁻ supports tumor cell growth through glutathione‐based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System x_c⁻ consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L‐type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single‐particle analyses of the xCT‐CD98hc complex. As wild‐type xCT‐CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild‐type, and enabled the cryoelectron microscopy (cryo‐EM) map to be obtained at 6.2 Å resolution by single‐particle analysis. The cryo‐EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus‐based strategy could represent a good choice toward solving unstable protein structures.

中文翻译：

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共识诱变方法提高了系统xc-转运蛋白xCT的热稳定性，并支持cryo-EM分析

系x _Ç ^-是一种氨基酸逆向转运该进口L-胱氨酸到细胞和细胞内的出口L-谷氨酸，以1：1的比例。由于L-胱氨酸为谷胱甘肽合成，系x必不可少的前体_Ç ^-通过基于谷胱甘肽氧化胁迫抗性的载体肿瘤细胞生长和被认为是用于癌症治疗的潜在的治疗靶。系统X _Ç ^-由两个亚基组成：轻链亚基SLC7A11（xCT）和重链亚基SLC3A2（也称为CD98hc或4F2hc），它们通过保守的二硫键连接。尽管另一个SLC7成员的最新结构，即L型氨基酸转运蛋白1（LAT1）与CD98hc的复合物，为理解氨基酸转运机理提供了结构基础，但是xCT的详细分子机理仍然未知。为了揭示分子机制，我们对xCT-CD98hc复合物进行了单颗粒分析。由于野生型xCT-CD98hc表现出较差的稳定性，无法将其纯化至同质，因此我们对xCT应用了共识诱变方法。与野生型相比，共有突变体显示出更高的稳定性，并使得能够在6点获得冷冻电子显微镜（cryo-EM）图。通过单颗粒分析获得2Å分辨率。低温电磁图显示足够的电子密度来分配二级结构。在xCT结构中，hash和arm域得到很好的解析，而bundle域则显示出一定的灵活性。CD98hc位于xCT跨膜结构域旁边。这项研究提供了xCT的结构基础，我们基于共识的策略可能是解决不稳定蛋白质结构的不错选择。