Properties of an Inward-Facing State of LeuT: Conformational Stability and Substrate Release
Properties of an Inward-Facing State of LeuT: Conformational Stability and Substrate Release
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DOI：
10.1016/j.bpj.2015.02.010
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发表时间：
2015-03-24
影响因子：
3.4
通讯作者：
Schiott, Birgit
中科院分区：
文献类型：
--
作者：
Grouleff, Julie;Sondergaard, Siri;Schiott, Birgit
亮氨酸转运蛋白（LeuT）是人类单胺转运蛋白的细菌同源物，是重要的药物靶点。目前还没有高分辨率的人类转运蛋白结构;然而，LeuT已经结晶在几个不同的构象状态。最近，LeuT的内向构象被解决，揭示了跨膜螺旋1a（TM 1a）的意外大的运动。我们已经进行了分子动力学模拟的突变和野生型转运蛋白，有和没有共结晶的Fab抗体片段，调查的性质，这种面向内的构象在膜环境中的运输LeuT。在所有的模拟，局部构象变化相对于晶体结构的一致性观察，特别是在TM 1a。伞式采样揭示了TM 1a倾斜的软潜力。此外，模拟面向内的LeuT与Na+离子和底物结合表明，其中一个Na+离子结合位点被完全破坏。释放丙氨酸和第二个Na+离子也观察到，洞察到的最后阶段的易位过程中的原子细节。
The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na+ ions and substrate bound suggest that one of the Na+ ion binding sites is fully disrupted. Release of alanine and the second Na+ ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.