The L-arginine/agmatine transporter AdiC is part of the arginine-dependent extreme acid resistance system of the bacterium Escherichia coli and its pathogenic varieties such as strain E. coli O157:H7. At the present time, there is a lack of knowledge concerning the role of water molecules and networks for the structure and function of AdiC, and solute transporters in general. The structure of the L-arginine/agmatine transporter AdiC was determined at 1.7 Å resolution by X-ray crystallography. This high resolution allowed for the identification of numerous water molecules buried in the structure. In combination with molecular dynamics (MD) simulations, we demonstrate that water molecules play an important role for stabilizing the protein and key residues, and act as placeholders for atoms of the AdiC substrates L-arginine and agmatine. MD simulations unveiled flexibility and restrained mobility of gating residues W202 and W293, respectively. Furthermore, a water-filled cavity was identified at the dimer interface of AdiC. The two monomers formed bridging interactions through water-mediated hydrogen bonds. The accessibility and presence of water molecules in this cavity was confirmed with MD simulations. Point mutations disrupting the interfacial water network validated the importance of water molecules for dimer stabilization. This work gives new insights into the role and importance of water molecules in the L-arginine/agmatine transporter AdiC for protein stabilization and substrate-binding site shaping and as placeholders of substrate atoms. Furthermore, and based on the observed flexibility and restrained mobility of gating residues, a mechanistic role of the gate flexibility in the transport cycle was proposed. Finally, we identified a water-filled cavity at the dimeric interface that contributes to the stability of the amino acid transporter oligomer.

中文翻译：

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氨基酸转运蛋白 AdiC 的高分辨率结构揭示了水分子和网络在寡聚化和底物结合中的作用

L-精氨酸/胍丁胺转运蛋白 AdiC 是大肠杆菌及其致病品种如大肠杆菌 O157:H7 的精氨酸依赖性极酸性抗性系统的一部分。目前，缺乏关于水分子和网络对 AdiC 的结构和功能以及一般溶质转运蛋白的作用的知识。L-精氨酸/胍丁胺转运蛋白 AdiC 的结构通过 X 射线晶体学以 1.7 Å 的分辨率确定。这种高分辨率可以识别埋在结构中的大量水分子。结合分子动力学 (MD) 模拟，我们证明水分子在稳定蛋白质和关键残基方面发挥着重要作用，并充当 AdiC 底物 L-精氨酸和胍丁胺的原子的占位符。MD 模拟分别揭示了门控残基 W202 和 W293 的灵活性和受限迁移率。此外，在 AdiC 的二聚体界面处发现了一个充满水的空腔。这两种单体通过水介导的氢键形成桥接相互作用。MD 模拟证实了该空腔中水分子的可及性和存在性。破坏界面水网络的点突变验证了水分子对二聚体稳定的重要性。这项工作为水分子在 L-精氨酸/胍丁胺转运蛋白 AdiC 中的作用和重要性提供了新的见解，用于蛋白质稳定和底物结合位点成形以及作为底物原子的占位符。此外，基于观察到的门控残基的灵活性和受限的移动性，提出了门灵活性在运输周期中的机械作用。最后，我们在二聚体界面发现了一个充满水的空腔，它有助于氨基酸转运蛋白寡聚体的稳定性。