兴奋性氨基酸转运体 3 底物识别的结构基础

Biao Qiu, Olga Boudker
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引用次数: 0

摘要

兴奋性氨基酸转运体(EAATs)位于细胞表面,利用离子梯度吸收 L-谷氨酸、L-天门冬氨酸和 D-天门冬氨酸等底物。在五种 EAATs 中,EAAT3 是唯一能有效转运 L-半胱氨酸(谷胱甘肽合成的底物)的同工酶。最近的研究表明,EAAT3 还能转运副代谢物 R-2-羟基戊二酸(R-2HG)。在这里,我们通过测定与不同底物结合的 EAAT3 的冷冻电镜结构,研究了底物杂交的结构基础。我们发现,L-半胱氨酸以硫酸盐形式与 EAAT3 结合,EAAT3 通过微调配位残基的局部构象来识别不同的底物。然而,使用纯化的人 EAAT3,我们无法观察到 R-2HG 的结合或转运。对与 L-半胱氨酸结合的 EAAT3 的成像显示了几种构象状态,包括门半开的外向状态和中断的钠结合位点。这些结构表明,在与底物结合后,门会完全关闭,并与最后一个钠离子结合。此外,我们还观察到,不同的底物会影响转运体在完全外向构象和通向内向构象的中间闭锁状态之间的分布,这表明转运速率与底物有关。
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Structural basis of the excitatory amino acid transporter 3 substrate recognition
Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.
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