Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.
Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.
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DOI：
10.3389/fmicb.2018.00831
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发表时间：
2018
影响因子：
5.2
通讯作者：
Yang C
中科院分区：
文献类型：
--
作者：
Yang Z;Meng Y;Zhao Q;Cheng B;Xu P;Yang C
NhaD型反向转运蛋白主要分布于各种变形菌中，特别是在海洋微生物和人类病原体中。这种分布以及这些菌株的致病特性表明，这些反向转运蛋白有助于调节高血糖调节，是潜在的药物靶点。两个NhaD同系物，NhaD 1和NhaD 2，从耐盐和嗜碱盐单胞菌属Y2，表现出相似的，高的体外活性，但在体内功能显着不同。为了寻找参与这些生理功能差异的关键结构域或残基，产生了由NhaD 1和NhaD 2片段组成的各种嵌合体。发现在残基1-67和464-492处的两个区域负责NhaD 2的稳健的体内功能，并且区域464-492对于反向转运蛋白的pH响应也是至关重要的。特别地，KNabc/N463 r的活性完全消除，KNabc/N463 r-C7嵌合体的活性高度恢复而离子抗性非常弱地恢复，这表明跨膜螺旋（TM）XIII对于NhaD 2的稳健离子抗性是至关重要的。使用定点突变，在TM XIII中的7个疏水残基被确定为NhaD 2离子易位的关键残基。与野生型NhaD 2的荧光共振能量转移（FRET）谱相比，N463 r嵌合体FRET效率的降低为N463 r融合蛋白的构象变化提供了有力证据，从而验证了TM XIII在NhaD 2的pH激活和生理功能中的结构功能。
NhaD-type antiporters are mainly distributed in various Proteobacteria, especially in marine microorganisms and human pathogens. This distribution as well as the pathogenic properties of these strains suggest that these antiporters contribute to the regulation of high osmoregulation and are potential drug targets. Two NhaD homologs, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. Y2 exhibits similar, high in vitro activity, but remarkably different in vivo functions. To search for critical domains or residues involved in these differences of physiological functions, various chimeras composed of NhaD1 and NhaD2 segments were generated. Two regions at residues 1–67 and 464–492 were found to be responsible for the robust in vivo function of NhaD2, and region 464–492 is also crucial to the pH response of the antiporter. In particular, the completely abolished activity of KNabc/N463r, highly recovered activity while very weakly recovered ion resistance of the KNabc/N463r-C7 chimera, suggested that transmembrane helix (TM) XIII is crucial for the robust ion resistance of NhaD2. Using site-directed mutagenesis, seven hydrophobic residues in TM XIII were identified as key residues for the ion translocation of NhaD2. Compared with the fluorescence resonance energy transfer (FRET) profile in the wild-type NhaD2, the reduced FRET efficiency of N463r chimeras provided solid evidence for conformational changes in the N463r fusion protein and consequently verified the structural functions of TM XIII in the pH activation and physiological functions of NhaD2.