Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012, Bern, Switzerland.
DS3Lab, System Group, Department of Computer Sciences, ETH Zurich, CH-8093, Zürich, Switzerland.
BMC Biol. 2021 Aug 30;19(1):179. doi: 10.1186/s12915-021-01102-4.
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.
L-精氨酸/胍丁胺转运蛋白 AdiC 是细菌大肠杆菌及其致病性变体（如大肠杆菌 O157:H7 菌株）中依赖精氨酸的极端酸抗性系统的一部分。目前，对于 AdiC 结构和功能以及一般溶质转运蛋白的水分子和网络的作用知之甚少。
通过 X 射线晶体学确定了 L-精氨酸/胍丁胺转运蛋白 AdiC 的结构，分辨率为 1.7Å。高分辨率允许鉴定出许多埋藏在结构中的水分子。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起着重要作用，并作为 AdiC 底物 L-精氨酸和胍丁胺原子的占位符。MD 模拟揭示了门控残基 W202 和 W293 的灵活性和受限的流动性。此外，在 AdiC 的二聚体界面处鉴定到一个充满水的空腔。两个单体通过水分子介导的氢键形成桥接相互作用。MD 模拟证实了该腔的可及性和水分子的存在。破坏界面水分子网络的点突变验证了水分子对二聚体稳定的重要性。
这项工作深入了解了水分子在 L-精氨酸/胍丁胺转运蛋白 AdiC 中的作用和重要性，对于蛋白质稳定和底物结合位点的形成以及作为底物原子的占位符具有重要意义。此外，基于观察到的门控残基的灵活性和受限的流动性，提出了门控灵活性在转运周期中的机械作用。最后，我们在二聚体界面处鉴定到一个充满水的空腔，该空腔有助于氨基酸转运蛋白寡聚物的稳定性。
