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首页> 外文期刊>The FEBS journal >Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion
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Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

机译：水通道蛋白NPA序列签名基序中阳离子排除的天冬酰胺的要求

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摘要

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region - in particular, the Asn position - is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.
机译：两个高度保守的NPA基序是水通道蛋白（AQP）家族的标志。 NPA三联体形成N末端螺旋加帽结构，Asn侧链位于水或溶质传导通道的中心，并被认为在AQP选择性中起重要作用。尽管通过突变分析已很好地表征了另一个AQP选择性过滤位点，即芳族/ Arg（ar / R）缩颈，但缺少有关NPA区-特别是Asn位置的实验数据。在这里，我们报告了一种新型的携带一个SPA主题而不是来自囊性纤维化患者的流行病原体伯克霍尔德森cenocepacia的NPA主题的SPA甘油糖蛋白的克隆和突变分析。在RefSeq中保存的1357个AQP序列中，我们仅鉴定出15个具有Asn交换的序列。使用直接和表型通透性检测，我们发现Asn和Ser在两个NPA位点均可自由互换，而不会影响蛋白质表达或水，甘油和甲胺的通透性。但是，NPA区中的其他突变导致通透性降低（S186C和S186D），无功能通道（N64D）甚至缺乏蛋白质表达（S186A和S186T）。使用电生理学，我们发现一个类似的哺乳动物AQP1 N76S突变体排除了质子和钾离子，但漏出了钠离子，这为Asn胜过其他氨基酸提供了依据。我们得出的结论是，在NPA图案的第一个位置上，只有Asn提供有效的螺旋帽稳定和阳离子排斥，而其他小的残基会损害结构稳定性或阳离子排斥，但不一定会损害水和溶质的渗透性。

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