Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2
Functional Interaction between the N and C Termini of NhaD Antiporters from Halomonas sp. Strain Y2
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DOI：
10.1128/jb.00302-17
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发表时间：
2017-05
影响因子：
3.2
通讯作者：
Yiwei Meng;Zhou Yang;Bin Cheng;Xinyu Nie;Shannan Li;Huijia Yin;P. Xu;Chunyu Yang
中科院分区：
文献类型：
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作者：
Yiwei Meng;Zhou Yang;Bin Cheng;Xinyu Nie;Shannan Li;Huijia Yin;P. Xu;Chunyu Yang
摘要耐盐嗜碱盐单胞菌Y2菌株的两个NhaD型反向转运蛋白NhaD 1和NhaD 2具有高度的序列同源性，但在Na+和Li+抗性方面表现出不同的生理功能。在本研究中，从NhaD 2的额外的4个C-末端残基的截短或这些蛋白质之间的39个N-末端残基的交换导致反向转运蛋白活性的完全丧失。有趣的是，组合NhaD 2的39个N-末端残基和7个C-末端残基（N39 D2-C7）部分恢复了Na+和Li+排出的活性，以及暴露于300 mM Na+和100 mM Li+胁迫后的互补生长。嵌合体N39 D2-C7的恢复的活性表明N和C末端在结构上彼此依赖并且协同地起作用。此外，荧光共振能量转移（FRET）分析表明，N和C末端相对接近，这可能是它们在离子转运中的协同作用。在N39 D2-C7的N-末端区域，用Pro取代Glu 38，使恢复的互补和转运活性消失。此外，NhaD 2中的这种氨基酸取代导致大肠杆菌KNabc中的互补能力急剧降低（与NhaD 1的水平相同），以及活性降低和pH曲线改变。关于NhaD反向转运蛋白的有限信息支持这些反向转运蛋白对于抵抗高盐度和碱度很重要的推测。此外，只有少数功能残基已被确定在NhaD反向转运蛋白，有有限的文献NhaD反向转运蛋白活性的分子机制。在这项研究中的嵌合体和突变体的反向转运能力的改变牵连的N和C末端，特别是Glu 38，在pH调节和离子易位的功能，最重要的是，这个带负电荷的残基在维持NhaD 2的生理功能的重要作用。这些发现进一步加深了我们对NhaD反向转运蛋白离子转运的分子机制的理解。
ABSTRACT Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile. IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.