Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

磷酸盐是多种细胞中不可或缺的代谢物，参与核苷酸和脂质合成、信号传导和化学能量储存。主要促进剂家族中的质子偶联磷酸盐转运蛋白对于植物和真菌的磷酸盐吸收至关重要。在导致人类疾病的不同原生动物寄生虫、对磷酸盐需求增加的乳腺癌细胞、骨重吸收过程中的破骨细胞样细胞以及用于磷酸盐稳态的人肠道 Caco2BBE 细胞中，都发现了类似的质子偶联磷酸盐转运蛋白。然而，质子驱动磷酸盐转运的机制仍不清楚。在这里，我们展示了来自Piriformospora indica的真核、高亲和力磷酸盐转运蛋白(PiPT) 天冬氨酸 324 (D324) 的去质子化触发磷酸盐释放。结合自由能采样的量子力学/分子力学分子动力学模拟已被用于识别 D324 从封闭结构转变为向内开放结构和磷酸盐释放的质子传输途径。然后通过诱变实验对 D45N 和 D45E 氨基酸取代的研究证实了如此获得的计算见解。我们的研究结果证实了结构预测的细胞质质子出口隧道的功能，并提出了对可滴定磷酸盐底物作用的见解。