Molecular Neuropharmacology and Genetics Laboratory, Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Maersk Tower 7-5, DK-2200, Copenhagen N, Denmark.
Membrane Protein Structural Biology Group, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Maersk Tower 7-9, DK-2200, Copenhagen N, Denmark.
Nat Commun. 2020 Feb 21;11(1):1005. doi: 10.1038/s41467-020-14735-w.
Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.
钠协同转运蛋白（NSS）在从细菌到人之间是保守的，它们是药物的靶点，包括抗抑郁药和精神兴奋剂。在这里，我们报告了原核 NSS 成员 LeuT 的 X 射线结构，该结构处于 Na/底物结合、内向封闭构象。为了获得这个结构，我们受到单分子荧光光谱和分子动力学模拟结果的指导，这些结果表明 L-苯丙氨酸结合和保守的 N 端色氨酸到丙氨酸的突变都促进了内向构象。与外向封闭构象相比，我们的结构揭示了跨膜片段（TM）5 的细胞质末端的主要倾斜，这与 N 端的释放但没有 TM1 的偶联运动一起，朝向第二个 Na 结合位点打开了一个宽阔的空腔。在其他 NSS 结构的背景下，这种 LeuT 转运循环关键中间产物的结构导致了 NSS 蛋白中底物和离子的细胞内释放机制的提出。
