A pathway leading to a cation-binding pocket determines the selectivity of the NhaP2 antiporter in Vibrio cholerae1.
A pathway leading to a cation-binding pocket determines the selectivity of the NhaP2 antiporter in Vibrio cholerae1.
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通往阳离子结合袋的途径决定了霍乱弧菌 1 中 NhaP2 反向转运蛋白的选择性。
DOI：
10.1139/bcb-2018-0146
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Dibrov,P
中科院分区：
文献类型：
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作者：
Mourin,M;Wai,A;O'Neil,J;Schubiger,CB;Häse,CC;Hausner,G;Dibrov,P
霍乱弧菌的Vc-NhaP 2反向转运蛋白可将H+交换为K+或Na+，但不交换较小的Li+。利用Phyre 2和Rosetta软件对Vc-NhaP 2进行结构模型的构建，并对该结构进行了初步的分析。所获得的模型表明，来自不同跨膜区段（TMS）的残基簇在膜中间形成推定的阳离子结合口袋：TMS V的D133和T132以及TMS VI的D162和E157。该模型还表明，来自TMS IX的L257、G258和N259与来自TMS X的T276、D273、Q280和Y251以及来自TMS XII的L289和L342一起形成易位离子的跨膜途径，其中内置过滤器确定阳离子选择性。丙氨酸扫描诱变鉴定的残基验证了该模型，表明结构修饰的途径导致改变阳离子选择性和运输活性。特别是，L257 A，G258 A，Q280 A和Y251 A变体获得了Li+/H+反向转运能力，这在未突变的反向转运蛋白中是不存在的。T276 A、D273 A和L289 A变体仅将K+交换为H+，而L342 A变体仅介导Na+/H+交换，从而保持严格的碱金属阳离子选择性。
The Vc-NhaP2 antiporter fromVibrio choleraeexchanges H+for K+or Na+but not for the smaller Li+. The molecular basis of this unusual selectivity remains unknown.Phyre2and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li+/H+antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K+for H+, while a L342A variant mediated Na+/H+exchange only, thus maintaining strict alkali cation selectivity.