Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.
Computational Biology, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
FASEB J. 2019 Feb;33(2):1989-1999. doi: 10.1096/fj.201800782R. Epub 2018 Sep 13.
The movement of ammonium across biologic membranes is a fundamental process in all living organisms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus protein (Amt/Mep/Rh) family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter AmtB specifically binds 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Upon POPG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted, to our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.
铵跨生物膜的运动是所有生物体中的一个基本过程，由普遍存在的铵转运体/甲基铵渗透酶/恒河猴蛋白（Amt/Mep/Rh）家族转运体介导。最近的结构分析和耦合质谱研究表明，大肠杆菌铵转运体 AmtB 特异性结合 1-棕榈酰-2-油酰磷脂酰甘油（POPG）。在 POPG 结合后，AmtB 的几个残基发生小的构象变化，使蛋白稳定，防止展开。然而，据我们所知，迄今为止还没有研究探索 POPG 与 AmtB 的结合是否具有功能后果。在这里，我们使用纯化成分的体外实验测定，以及分子动力学模拟，来描述 POPG 结合与 AmtB 活性之间的关系。我们表明 AmtB 活性是电致的。我们的结果表明，在古细菌和真细菌中，Amt 的活性在分子水平上可能不同。我们还表明，POPG 是 AmtB 活性的重要辅助因子，并且在没有 POPG 的情况下，AmtB 无法完成完整的转运循环。此外，我们的模拟揭示了在脂质双层的胞内侧 AmtB 亚基之间以前未发现的 POPG 结合位点。讨论了可能的分子机制，以解释 POPG 的功能作用。-Mirandela，G. D.，Tamburrino，G.，Hoskisson，P. A.，Zachariae，U.，Javelle，A. 脂质环境决定了大肠杆菌铵转运体 AmtB 的活性。
