Vinothkumar Kutti R, Smits Sander H J, Kühlbrandt Werner
Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
EMBO J. 2005 Aug 3;24(15):2720-9. doi: 10.1038/sj.emboj.7600727. Epub 2005 Jul 14.
Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85 degrees C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 A projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and an approximately 2 A shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter.
钠氢反向转运蛋白是一种依赖于pH值的膜转运蛋白，可维持活细胞内氢离子和钠离子的稳态。来自嗜热栖热菌（一种在85摄氏度下生长最佳的嗜热古菌）的MjNhaP1被克隆并在大肠杆菌中表达。在pH值为4的条件下，从纯化的蛋白质中获得了二维晶体。电子冷冻显微镜产生了一个8埃的投影图。与相关的大肠杆菌反向转运蛋白NhaA一样，MjNhaP1是一种二聚体，但除此之外，这两种反向转运蛋白的结构有显著差异。MjNhaP1的图谱显示，在二聚体中心有细长的密度，在二聚体核心两侧有一簇密度峰，表明有一束4-6个跨膜螺旋。原位研究了pH值对MjNhaP1结构的影响。在pH值为6及以上时，螺旋束内的密度分布发生了重大变化，螺旋束相对于二聚体核心的位置发生了约2埃的移动。低pH值和高pH值下的两种构象很可能分别代表反向转运蛋白的关闭状态和开放状态。
