Department of Physiology, University of California, Los Angeles, Los Angeles, California 90095-1759, USA.
Nature. 2010 Dec 16;468(7326):988-91. doi: 10.1038/nature09580. Epub 2010 Dec 5.
Membrane co-transport proteins that use a five-helix inverted repeat motif have recently emerged as one of the largest structural classes of secondary active transporters. However, despite many structural advances there is no clear evidence of how ion and substrate transport are coupled. Here we report a comprehensive study of the sodium/galactose transporter from Vibrio parahaemolyticus (vSGLT), consisting of molecular dynamics simulations, biochemical characterization and a new crystal structure of the inward-open conformation at a resolution of 2.7 Å. Our data show that sodium exit causes a reorientation of transmembrane helix 1 that opens an inner gate required for substrate exit, and also triggers minor rigid-body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose readily to enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium-binding sites, the unwound portion of transmembrane helix 1 and the substrate-binding site that is essential in the coupling of co-transport.
最近，利用五螺旋倒位重复基序的膜共转运蛋白已成为最大的结构类别的次级主动转运蛋白之一。然而，尽管有许多结构上的进展，但对于离子和底物的转运如何偶联仍没有明确的证据。在这里，我们报告了对副溶血弧菌（vSGLT）的钠/半乳糖转运蛋白的全面研究，包括分子动力学模拟、生化特性分析和一个分辨率为 2.7 Å 的新的内向开放构象的晶体结构。我们的数据表明，钠离子的外排导致跨膜螺旋 1的重排，打开了底物外排所需的内门，并且还引发了两组跨膜螺旋束的微小刚体运动。这一连串的事件，由钠离子的释放引发，确保了离子和底物释放的适当时间。一旦开始运动，这些分子变化就会削弱转运蛋白与底物的结合，使半乳糖能够轻易地进入细胞内空间。此外，我们还确定了钠结合位点、跨膜螺旋 1的未缠绕部分和底物结合位点之间的变构途径，这对于共转运的偶联是必不可少的。
