Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter:: Structural and functional implications
Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter:: Structural and functional implications
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DOI：
10.1016/j.jmb.2008.03.067
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发表时间：
2008-06-06
影响因子：
5.6
通讯作者：
Padan, Etana
中科院分区：
文献类型：
--
作者：
Rimon, Abraham;Hunte, Carola;Padan, Etana
最近确定的晶体结构的NhaA，Na+/H+反向转运蛋白的大肠杆菌，表明，以前构建的一系列NhaA-碱性磷酸酶（PhoA）融合正确预测的拓扑结构NhaA的12个跨膜段（TMS），与C-和N-末端指向细胞质。在这里，我们表明，这些NhaA-PhoA融合提供了一个很好的工具，映射表位的三个NhaA特异性构象单克隆抗体（mAb），其中两个显着抑制反向转运蛋白。通过鉴定哪种NhaA融合体被相应的mAb结合，表位被定位到NhaA的小片段。然后通过靶向Cys扫描诱变结合化学修饰进行精确定位。最有趣的是，在环X-XI（细胞质）和环XI-XII（周质）中鉴定了抑制性mAb 5 H4和2C 5的表位，所述环X-XI和环XI-XII分别在膜的细胞质侧和周质侧上通过TMS XI连接。所揭示的mAb的位置表明mAb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mAb 6179与NhaA的功能性PKC末端结合。(c)2008爱思唯尔有限公司保留所有权利。
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 6179 binds to the functionally dispensable C-terminus of NhaA. (c) 2008 Elsevier Ltd. All rights reserved.