Lu Muxue, Yang Guangzhe, Li Peifang, Wang Zhigang, Fu Shan, Zhang Xiang, Chen Xi, Shi Mingxing, Ming Zhenhua, Xia Jixing
State Key Laboratory of Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, Nanning, China.
Front Plant Sci. 2018 May 7;9:606. doi: 10.3389/fpls.2018.00606. eCollection 2018.
Nrat1 is a member of the natural resistance-associated macrophage protein (Nramp) family of metal ion transporters in all organisms. Different from other Nramp members capable of transporting divalent metals, Nrat1 specifically transports trivalent aluminum (Al) ion. However, molecular mechanism underlying the Al transport selectivity of Nrat1 remains unknown. Here, we performed structure-function analyses of Nrat1 and other Nramp members to gain insights into the determinants of ion selectivity. A phylogenetic analysis showed that plant Nramp transporters could be divided into five groups. OsNrat1 was found in one of the individual clades and clustered with SbNrat1 and ZmNrat1 on the evolutionary tree. Structural modeling revealed that Nrat1 transporters adopted a common LeuT fold shared by many Nramp-family transporters that likely employed an identical transport mechanism. Sequence alignment and evolutionary conservation analysis of amino acids identified a metal-permeation pathway of Nrat1 centered at the metal binding site. The metal binding site of Nrat1 was characterized by two conserved sequence motifs, i.e., the Asp-Pro-Ser-Asn motif (motif A) and the Ala-Ile-Ile-Thr motif (motif B). Replacement of the Ala-Met-Val-Met motif B of the OsNramp3 manganese (Mn) transporter to that of Nrat1 resulted in a partial gain of Al transport activity and a total loss of Mn in yeast. Conversely, substitution of the motif B of OsNrat1 with that of OsNramp3 altered the Al transport activity. These observations indicated the metal binding site, particularly the motif B, as a key determinant of Al selectivity of Nrat1.
Nrat1是所有生物体中金属离子转运蛋白的天然抗性相关巨噬细胞蛋白（Nramp）家族的成员。与其他能够转运二价金属的Nramp成员不同，Nrat1特异性转运三价铝（Al）离子。然而，Nrat1对Al转运选择性的分子机制仍然未知。在这里，我们对Nrat1和其他Nramp成员进行了结构功能分析，以深入了解离子选择性的决定因素。系统发育分析表明，植物Nramp转运蛋白可分为五组。在其中一个单独的进化枝中发现了OsNrat1，并且在进化树上与SbNrat1和ZmNrat1聚类。结构建模显示，Nrat1转运蛋白采用了许多Nramp家族转运蛋白共有的常见LeuT折叠，可能采用相同的转运机制。氨基酸序列比对和进化保守性分析确定了以金属结合位点为中心的Nrat1的金属渗透途径。Nrat1的金属结合位点由两个保守的序列基序表征，即Asp-Pro-Ser-Asn基序（基序A）和Ala-Ile-Ile-Thr基序（基序B）。将OsNramp3锰（Mn）转运蛋白的Ala-Met-Val-Met基序B替换为Nrat1的基序B，导致酵母中Al转运活性部分增加，而Mn完全丧失。相反，用OsNramp3的基序B替换OsNrat1的基序B改变了Al转运活性。这些观察结果表明金属结合位点，特别是基序B，是Nrat1对Al选择性的关键决定因素。
