Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.
Proc Natl Acad Sci U S A. 2013 May 21;110(21):8489-94. doi: 10.1073/pnas.1221279110. Epub 2013 May 2.
Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.
钠离子转运体（NSSs）通过重摄取神经递质在信号转导中发挥关键作用。真核 NSSs 依赖氯离子，而类似于 LeuT 的原核 NSS 同源物则不依赖氯离子，但在真核 NSSs 具有丝氨酸的位置含有酸性残基（LeuT 中的Glu290）。LeuT-E290S 突变体显示氯离子依赖性活性。我们表明，在与溴化物或氯化物共结晶的 LeuT-E290S 中，阴离子由 Tyr47、Ser290 和 Thr254 的侧链羟基以及 Gln250 的侧链酰胺配位。结合在 Na1 位的阴离子和附近的钠离子通过连续的氢键网络将其配位残基与 LeuT 的细胞外门连接起来。结构提供的具体见解，结合底物结合研究和分子动力学模拟的结果，揭示了 NSS 的阴离子依赖性封闭机制，并阐明了氯离子结合的功能作用。
