Appel Matthias, Hizlan Dilem, Vinothkumar Kutti R, Ziegler Christine, Kühlbrandt Werner
Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, Frankfurt am Main, Germany.
J Mol Biol. 2009 May 8;388(3):659-72. doi: 10.1016/j.jmb.2009.03.010.
NhaA, the main sodium-proton exchanger in the inner membrane of Escherichia coli, regulates the cytosolic concentrations of H+ and Na+. It is inactive at acidic pH, becomes active between pH 6 and pH 7, and reaches maximum activity at pH 8. By cryo-electron microscopy of two-dimensional crystals grown at pH 4 and incubated at higher pH, we identified two sequential conformational changes in the protein in response to pH or substrate ions. The first change is induced by a rise in pH from 6 to 7 and marks the transition from the inactive state to the pH-activated state. pH activation, which precedes the ion-induced conformational change, is accompanied by an overall expansion of the NhaA monomer and a local ordering of the N-terminus. The second conformational change is induced by the substrate ions Na+ and Li+ at pH above 7 and involves a 7-A displacement of helix IVp. This movement would cause a charge imbalance at the ion-binding site that may trigger the release of the substrate ion and open a periplasmic exit channel.
NhaA是大肠杆菌内膜中的主要钠-质子交换体，可调节胞质中H⁺和Na⁺的浓度。它在酸性pH值下无活性，在pH 6至pH 7之间变得活跃，并在pH 8时达到最大活性。通过对在pH 4下生长并在较高pH值下孵育的二维晶体进行冷冻电子显微镜观察，我们确定了蛋白质响应pH值或底物离子的两个连续构象变化。第一个变化是由pH从6升高到7引起的，标志着从无活性状态到pH激活状态的转变。在离子诱导的构象变化之前的pH激活伴随着NhaA单体的整体扩张和N端的局部有序化。第二个构象变化是由pH高于7时的底物离子Na⁺和Li⁺诱导的，涉及螺旋IVp的7 Å位移。这种移动会在离子结合位点引起电荷不平衡，这可能触发底物离子的释放并打开一个周质出口通道。
