Grouleff Julie, Søndergaard Siri, Koldsø Heidi, Schiøtt Birgit
Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark.
Center for Insoluble Protein Structures and Interdisciplinary Nanoscience Center, Department of Chemistry, Aarhus University, Aarhus, Denmark.
Biophys J. 2015 Mar 24;108(6):1390-1399. doi: 10.1016/j.bpj.2015.02.010.
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.
亮氨酸转运体（LeuT）是人类单胺转运体的细菌同源物，而人类单胺转运体是重要的药物靶点。目前尚无人类转运体的高分辨率结构；然而，LeuT已在几种不同的构象状态下结晶。最近，解析了LeuT的向内构象，揭示了跨膜螺旋1a（TM1a）出人意料的大幅移动。我们对突变型和野生型转运体进行了分子动力学模拟，有和没有共结晶的Fab抗体片段，以研究这种向内构象在膜环境中与LeuT转运相关的特性。在所有模拟中，均一致观察到相对于晶体结构的局部构象变化，尤其是在TM1a中。伞形抽样揭示了TM1a倾斜的软势垒。此外，对结合了Na(+)离子和底物的向内构象的LeuT进行的模拟表明，其中一个Na(+)离子结合位点完全被破坏。还观察到丙氨酸和第二个Na(+)离子的释放，从而在原子水平上详细洞察了转运过程的最后阶段。
