Sialin, a member of the solute carrier 17 (SLC17) transporter family, is unique in its ability to transport not only sialic acid using a pH-driven mechanism, but also transport mono and diacidic neurotransmitters, such as glutamate and N-acetylaspartylglutamate (NAAG), into synaptic vesicles via a membrane potential-driven mechanism. While most transporters utilize one of these mechanisms, the structural basis of how Sialin transports substrates using both remains unclear. Here, we present the cryogenic electron-microscopy structures of human Sialin: apo cytosol-open, apo lumen-open, NAAG–bound, and inhibitor–bound. Our structures show that a positively charged cytosol-open vestibule accommodates either NAAG or the Sialin inhibitor Fmoc-Leu-OH, while its luminal cavity potentially binds sialic acid. Moreover, functional analyses along with molecular dynamics simulations identify key residues in binding sialic acid and NAAG. Thus, our findings uncover the essential conformational states in NAAG and sialic acid transport, demonstrating a working model of SLC17 transporters.

Sialin 是溶质载体 17 (SLC17) 转运蛋白家族的一员，其独特之处在于它不仅能够利用 pH 驱动机制转运唾液酸，还能转运单酸和二酸神经递质，例如谷氨酸和 N-乙酰天冬氨酰谷氨酸 (NAAG) ），通过膜电位驱动机制进入突触小泡。虽然大多数转运蛋白利用其中一种机制，但唾液酸蛋白如何利用这两种机制转运底物的结构基础仍不清楚。在这里，我们展示了人唾液酸唾液酸蛋白的低温电子显微镜结构：apo 胞质开放、apo 腔开放、NAAG 结合和抑制剂结合。我们的结构表明，带正电荷的胞质开放前庭可容纳 NAAG 或唾液酸蛋白抑制剂 Fmoc-Leu-OH，而其腔可能与唾液酸结合。此外，功能分析和分子动力学模拟确定了唾液酸和 NAAG 结合的关键残基。因此，我们的研究结果揭示了 NAAG 和唾液酸转运中的基本构象状态，展示了 SLC17 转运蛋白的工作模型。