Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension.
Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension.
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DOI：
10.1016/j.jmb.2009.12.055
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发表时间：
2010-03-12
影响因子：
5.6
通讯作者：
Ben-Tal N
中科院分区：
文献类型：
--
作者：
Schushan M;Xiang M;Bogomiakov P;Padan E;Rao R;Ben-Tal N
人类NHA2是一种Na+/H+反向转运蛋白，最近与原发性高血压有关。我们使用一系列计算工具和进化守恒分析来建立并验证基于远亲细菌转运体NhaA的晶体结构的NHA2三维模型。该模型通过表型筛选对NHA2在酵母中的转运功能、离子选择性和pH依赖性进行了诱变评价。我们描述了一簇基本的，高度保守的可滴定残基，位于由两个不连续的倒置拓扑螺旋组成的组装区域，每个被延伸链中断。而在NhaA中，相反的带电残基补偿了该组装中产生的部分偶极子，在NHA2中，极性但不带电的残基就足够了。我们的发现导致了一个与众所周知的电中性NHE1和电致性NhaA亚型相比较的运输机制模型。本研究确立了NHA2作为最近在植物和后生动物中发现的知之甚少但普遍存在的CPA2反转运蛋白家族的原型，并阐明了一种结构驱动的方法来获取新发现转运蛋白的功能信息。
Human NHA2 is a poorly characterized Na+/H+ antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.
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