Stochastic steps in secondary active sugar transport
Stochastic steps in secondary active sugar transport
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DOI：
10.1073/pnas.1525378113
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发表时间：
2016-07-05
影响因子：
11.1
通讯作者：
Grabe, Michael
中科院分区：
文献类型：
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作者：
Adelman, Joshua L.;Ghezzi, Chiara;Grabe, Michael
二级活性转运蛋白，如采用亮氨酸转运蛋白折叠的转运蛋白，存在于生命的所有领域，它们具有利用离子梯度中储存的能量来积累生命所必需的小分子以及排出有毒和有害化合物的独特能力。这些蛋白质如何将离子结合和运输耦合到伴随的底物流是一个基本的结构和生物物理问题，随着不同结构状态下转运蛋白的高分辨率结构的出现，该问题开始在原子水平上得到回答。尽管如此，转运蛋白的动态特性，如离子/底物结合顺序以及结合如何引发构象变化，并没有从静态结构中揭示出来，但这对于理解它们的功能至关重要。在这里，我们报告了一系列对糖转运蛋白vSGLT进行的分子模拟，这些模拟有助于了解底物和离子如何从转运蛋白的内向状态释放出来。我们的模拟表明，释放的顺序是随机的。功能实验的目的是测试这一预测的人类同源物，hSGLT 1，我们还发现，细胞质释放是不有序的，但我们证实，从细胞外空间的底物和离子结合是有序的。我们的研究结果统一了关于离子和底物的细胞质释放的相互矛盾的已发表结果，并暗示了超家族中的其他转运蛋白可能缺乏离子和底物之间的协调的可能性。
Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.