Critical Involvement of the E373-D434 Region in the Acid Sensitivity of a NhaB-Type Na+/H+ Antiporter from Vibrio alginolyticus
Critical Involvement of the E373-D434 Region in the Acid Sensitivity of a NhaB-Type Na+/H+ Antiporter from Vibrio alginolyticus
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DOI：
10.1021/bi300738v
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发表时间：
2012-10-02
期刊：
影响因子：
2.9
通讯作者：
Yamaguchi, Toshio
中科院分区：
文献类型：
--
作者：
Kiriyama, Wakako;Nakamura, Tatsunosuke;Yamaguchi, Toshio
已经确定的是，VaNhaB，在溶藻弧菌中发现的NhaB型Na+/H+反向转运蛋白，表现出惊人的酸敏感性。然而，反向转运活性的pH依赖性调节机制的分子基础还有待研究。在这项研究中，我们产生了各种嵌合蛋白组成的VaNhaB和pH不敏感的直向同源物在大肠杆菌（EcNhaB）和分析的pH响应的Na+/H+反向转运活动，寻找关键的残基或结构域，参与的pH敏感性VaNhaB。我们的结果揭示了环8-环9区域（E373-D434）内的一段氨基酸残基对VaNhaB的酸敏感性的重要性，沿着可能涉及广泛分布在VaNhaB的一级结构中的其他未鉴定的残基。此外，我们证明了VaNhaB的E373-D434区域能够赋予我们的pH不敏感嵌合反向转运蛋白一定程度的酸敏感性，该嵌合反向转运蛋白主要由EcNhaB组成，除了在N-末端的7个氨基酸取代。这一结果有力地表明，E373-D434区域能够至少部分地作为在酸性pH下降低NhaB型反向转运蛋白活性的机制发挥作用。
It has been well established that VaNhaB, a NhaB-type Na+/H+ antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na+/H+ antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8- loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.