Sphingosine-1-phosphate (S1P) is an important signaling sphingolipid that regulates the immune system, angiogenesis, auditory function, and epithelial and endothelial barrier integrity. Spinster homolog 2 (Spns2) is an S1P transporter that exports S1P to initiate lipid signaling cascades. Modulating Spns2 activity can be beneficial in treatments of cancer, inflammation, and immune diseases. However, the transport mechanism of Spns2 and its inhibition remain unclear. Here, we present six cryo-EM structures of human Spns2 in lipid nanodiscs, including two functionally relevant intermediate conformations that link the inward- and outward-facing states, to reveal the structural basis of the S1P transport cycle. Functional analyses suggest that Spns2 exports S1P via facilitated diffusion, a mechanism distinct from other MFS lipid transporters. Finally, we show that the Spns2 inhibitor 16d attenuates the transport activity by locking Spns2 in the inward-facing state. Our work sheds light on Spns2-mediated S1P transport and aids the development of advanced Spns2 inhibitors.

1-磷酸鞘氨醇 (S1P) 是一种重要的信号传导鞘脂，可调节免疫系统、血管生成、听觉功能以及上皮和内皮屏障的完整性。 Spinster 同源物 2 (Spns2) 是一种 S1P 转运蛋白，可输出 S1P 以启动脂质信号级联反应。调节 Spns2 活性有助于治疗癌症、炎症和免疫疾病。然而，Spns2的转运机制及其抑制仍不清楚。在这里，我们展示了脂质纳米盘中人类 Spns2 的六种冷冻电镜结构，包括连接向内和向外状态的两个功能相关的中间构象，以揭示 S1P 运输循环的结构基础。功能分析表明 Spns2 通过促进扩散输出 S1P，这是一种与其他 MFS 脂质转运蛋白不同的机制。最后，我们发现 Spns2 抑制剂 16d 通过将 Spns2 锁定在向内状态来减弱转运活性。我们的工作揭示了 Spns2 介导的 S1P 转运，并有助于先进 Spns2 抑制剂的开发。