The divalent anion sodium symporter (DASS) family of transporters (SLC13 family in humans) are key regulators of metabolic homeostasis, disruption of which results in protection from diabetes and obesity, and inhibition of liver cancer cell proliferation. Thus, DASS transporter inhibitors are attractive targets in the treatment of chronic, age-related metabolic diseases. The characterisation of several DASS transporters has revealed variation in the substrate selectivity and flexibility in the coupling ion used to power transport. Here, using the model DASS co-transporter, VcINDY from Vibrio cholerae, we have examined the interplay of the three major interactions that occur during transport: the coupling ion, the substrate, and the lipid environment. Using a series of high-throughput thermostability-based interaction assays, we have shown that substrate binding is Na+-dependent; a requirement that is orchestrated through a combination of electrostatic attraction and Na+-induced priming of the binding site architecture. We have identified novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carboxylate groups in the ligand that are precisely distanced to satisfy carboxylate interaction regions of the substrate-binding site. We have also identified a complex relationship between substrate and lipid interactions, which suggests a dynamic, regulatory role for lipids in VcINDY's transport cycle.

中文翻译：

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基于热稳定性的结合分析揭示了细菌 DASS 转运蛋白 VcINDY 中阳离子、底物和脂质结合的复杂相互作用

二价阴离子钠同向转运蛋白 (DASS) 转运蛋白家族（人类的 SLC13 家族）是代谢稳态的关键调节剂，破坏代谢稳态可预防糖尿病和肥胖症，并抑制肝癌细胞增殖。因此，DASS 转运蛋白抑制剂是治疗慢性、年龄相关代谢疾病的有吸引力的靶点。几个 DASS 转运蛋白的表征揭示了用于驱动传输的耦合离子的底物选择性和灵活性的变化。在这里，我们使用来自霍乱弧菌的 DASS 协同转运蛋白模型 VcINDY，研究了转运过程中发生的三种主要相互作用的相互作用：耦合离子、底物和脂质环境。使用一系列基于高通量热稳定性的相互作用分析，我们已经证明底物结合是 Na+ 依赖性的；通过静电吸引和 Na+ 诱导的结合位点结构引发的组合精心设计的要求。我们已经确定了新的 DASS 配体，并揭示了配体结合取决于配体中两个羧酸根基团的要求，这些羧酸根基团的距离精确，以满足底物结合位点的羧酸根相互作用区域。我们还确定了底物和脂质相互作用之间的复杂关系，这表明脂质在 VcINDY 的运输循环中具有动态的调节作用。我们已经确定了新的 DASS 配体，并揭示了配体结合取决于配体中两个羧酸根基团的要求，这些羧酸根基团的距离精确，以满足底物结合位点的羧酸根相互作用区域。我们还确定了底物和脂质相互作用之间的复杂关系，这表明脂质在 VcINDY 的运输循环中具有动态的调节作用。我们已经确定了新的 DASS 配体，并揭示了配体结合取决于配体中两个羧酸根基团的要求，这些羧酸根基团的距离精确，以满足底物结合位点的羧酸根相互作用区域。我们还确定了底物和脂质相互作用之间的复杂关系，这表明脂质在 VcINDY 的运输循环中具有动态的调节作用。