Rimon Abraham, Hunte Carola, Michel Hartmut, Padan Etana
Department of Biochemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Mol Biol. 2008 Jun 6;379(3):471-81. doi: 10.1016/j.jmb.2008.03.067. Epub 2008 Apr 4.
The recently determined crystal structure of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.
最近确定的大肠杆菌钠离子/氢离子反向转运蛋白NhaA的晶体结构表明，先前构建的一系列NhaA-碱性磷酸酶（PhoA）融合蛋白正确预测了NhaA的12个跨膜片段（TMS）的拓扑结构，其C端和N端均指向细胞质。在此，我们表明这些NhaA-PhoA融合蛋白为定位三种NhaA特异性构象单克隆抗体（mAb）的表位提供了一个极好的工具，其中两种单克隆抗体能强烈抑制该反向转运蛋白。通过确定各自的单克隆抗体与哪种NhaA融合蛋白结合，表位被定位到NhaA的小片段区域。然后通过靶向半胱氨酸扫描诱变结合化学修饰进行精确映射。最有趣的是，抑制性单克隆抗体5H4和2C5的表位分别在环X-XI（细胞质）和环XI-XII（周质）中被确定，它们分别通过膜细胞质侧和周质侧的TMS XI相连。所揭示的单克隆抗体的位置表明，单克隆抗体的结合扭曲了独特的NhaA TMS IV/XI组装，从而抑制了NhaA的活性。非抑制性单克隆抗体6F9与NhaA功能上可有可无的C端结合。
