Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release.
Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release.
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DOI：
10.1371/journal.pcbi.1002246
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发表时间：
2011-10
影响因子：
4.3
通讯作者：
Schiøtt B
中科院分区：
文献类型：
--
作者：
Koldsø H;Noer P;Grouleff J;Autzen HE;Sinning S;Schiøtt B
单胺转运蛋白负责突触信号传导的终止，并参与抑郁、食欲控制和焦虑等神经过程。尽管进行了大量的研究工作，但单胺转运蛋白的结构以及离子和底物的转运机制仍然是未知的。人血清素转运体（hSERT）的结构知识是非常期待了解的机制的细节，底物易位和结合抗抑郁药和滥用药物。同源亮氨酸转运蛋白的晶体结构的公布导致了单胺转运蛋白的同源模型。在这里，我们提出了扩展的分子动力学模拟实验支持的同源性模型的hSERT与和没有天然底物产生的蛋白质二聚体的模拟总数超过1.5 µs。模拟揭示了hSERT从一个面向外的闭塞构象的一个面向内的构象在一个基板结合状态的过渡。模拟与第二基板在拟议的共转运效应网站没有导致与易位的构象变化。中心底物结合位点变得完全暴露于细胞质，从而使Na+-离子在Na 2-位点中，并且底物通过水相互作用与细胞质直接接触。模拟揭示了钠是如何释放的，并显示了早期底物运输事件的迹象。离子解离的Na 2-网站驱动易位的概念是支持的Na 2-网站突变体的实验研究。跨膜螺旋（TM）1和6被确定为参与运输过程中最大运动的螺旋。人5-羟色胺转运蛋白属于神经递质转运蛋白家族，其位于突触前神经末端，负责突触5-羟色胺信号传导的终止。5-羟色胺浓度的不平衡与各种神经元疾病有关，如抑郁症、食欲调节等。可获得的hSERT结构信息非常有限，但据信该蛋白质通过交替进入机制发挥作用，其中中心结合位点暴露于细胞外部或内部。我们以前已经发表了一个实验验证的hSERT向外闭塞的同源模型，在这里，我们揭示了面向内的构象的hSERT从分子动力学模拟，从中我们可以确定的主要运动发生在易位。从面向内的构象，我们观察到离子释放，揭示了重要的信息，在运输过程中的事件顺序。在钠离子转运后，底物也显示出早期转运事件。如实验所暗示的，离子遵循细胞质途径，并且配体结合位点通过该相同途径被水完全溶剂化。使用Asp 437 Asn突变体的hSERT的实验证实了Asp 437是控制离子转运的中心残基的预测。
Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport. The human serotonin transporter belongs to the family of neurotransmitter transporters, which are located in the presynaptic nerve end, from where it is responsible for termination of synaptic serotonin signaling. Imbalance in serotonin concentration is related to various neuronal conditions such as depression, regulation of appetite etc. Very limited structural information of hSERT is available, but it is believed that the protein functions through an alternating access mechanism, where the central binding site is either exposed to the outside or the inside of the cell. We have previously published an experimentally validated outward-occluded homology model of hSERT, and here we reveal the inward-facing conformation of hSERT from molecular dynamics simulations, from which we can identify the main movements occurring during the translocation. From the inward-facing conformation we observe ion release, revealing important information on the sequence of events during transport. Following transport of the sodium ion, the substrate also shows early events of transport. The ion follows a cytoplasmic pathway as hinted at from experiments, and the ligand binding site becomes fully solvated by water through this same pathway. Experiments using an Asp437Asn mutant of hSERT confirm the prediction that Asp437 is a central residue in controlling ion transport.
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影响因子：
2.9
作者：
Kroncke, Brett M.;Horanyi, Peter S.;Columbus, Linda
通讯作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.0804659105
发表时间：
2008-07-29
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Rudnick, Gary
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作者：
Kilic, F;Murphy, DL;Rudnick, G
通讯作者：
Rudnick, G
DOI：
10.1016/j.bbrc.2009.01.128
发表时间：
2009-03-20
影响因子：
3.1
作者：
Fjorback, Anja W.;Pla, Patrick;Nyengaard, Jens R.
通讯作者：
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