Mechanism for alternating access in neurotransmitter transporters
Mechanism for alternating access in neurotransmitter transporters
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DOI：
10.1073/pnas.0804659105
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发表时间：
2008-07-29
影响因子：
11.1
通讯作者：
Rudnick, Gary
中科院分区：
文献类型：
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作者：
Forrest, Lucy R.;Zhang, Yuan-Wei;Rudnick, Gary
LeuT是哺乳动物神经递质转运蛋白的细菌同系物，其晶体结构显示结合底物的分子基本上暴露于细胞外空间，但从细胞质中闭塞。因此，必须存在一个替代构象的LeuT，其中底物是可访问的细胞质和相应的机制，开关的可访问性从一侧的膜到其他。在这里，我们确定了哺乳动物血清素转运蛋白（SERT）（LeuT相同的转运蛋白家族的成员）的交替构象的细胞质可及性途径。我们还提出了一个模型的胞质面对的状态，利用内部pseuclosymmetry中观察到的晶体结构。LeuT包含两个结构相似的重复序列（TMs 1 -5和TMs 6-10），它们相对于膜平面是反向的。它们之间的构象差异导致细胞外途径的形成。我们的模型为面向细胞质的状态交换的两个重复的构象，从而暴露的基板和离子结合位点的细胞质。连接两种状态的构象变化主要涉及由跨膜螺旋1、2、6和7组成的4螺旋束的倾斜。改变该束的倾斜角基本上等同于改变两个重复序列的构象。广泛的SERT诱变和可访问性测量，使用半胱氨酸试剂，容纳我们的模型。这些观察结果可能与其他转运蛋白家族有关，其中许多家族含有内部反向重复序列。
Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseuclosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1-5 and TMs 6-10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.