Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1
Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1
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DOI：
10.1111/j.1742-4658.2012.08613.x
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发表时间：
2012-07-01
期刊：
影响因子：
5.4
通讯作者：
Maeshima, Masayoshi
中科院分区：
文献类型：
--
作者：
Kawachi, Miki;Kobae, Yoshihiro;Maeshima, Masayoshi
拟南芥MTP1是阳离子扩散促进剂家族的一种液泡膜Zn2+/H+逆向转运蛋白。在此，我们通过对金属敏感酵母菌株中50多种突变变体进行功能互补试验，对AtMTP1介导的转运及其显著的Zn2+选择性进行了结构 - 功能分析。这与基于大肠杆菌广谱二价阳离子转运蛋白YiiP晶体结构的AtMTP1同源建模相结合。大肠杆菌YiiP在细胞质C末端的Zn2+结合位点以及由跨膜螺旋TM2和TM5形成的孔在AtMTP1中是保守的。尽管在大肠杆菌YiiP中不存在，但AtMTP1延伸的N末端胞质结构域中的Cys31和Cys36对于锌敏感酵母菌株的互补是必需的。在跨膜孔内活性Zn2+结合位点的胞质侧，TM5中的Asn258或TM2中的Ser101被丙氨酸替代非选择性地增强了AtMTP1赋予的金属耐受性。建模预测这些残基阻碍胞质Zn2+进入AtMTP1的膜内Zn2+结合位点。紧邻的富含组氨酸的胞质环的构象变化可能会使Asn258移位并允许Zn2+进入孔中。这将使Zn2+转运与富含组氨酸的环动态耦合，从而作为细胞质Zn2+水平的选择性过滤器或传感器。AtMTP1内不同位点的单个突变赋予酵母对钴和镉的耐受性，包括N末端和富含组氨酸的分子内胞质结构域的缺失，以及跨膜孔两侧或参与分子内或分子间结构域相互作用的单个残基的突变，所有这些在非选择性的大肠杆菌YiiP中都不保守。
Arabidopsis thaliana MTP1 is a vacuolar membrane Zn2+/H+ antiporter of the cation diffusion facilitator family. Here we present a structurefunction analysis of AtMTP1-mediated transport and its remarkable Zn2+ selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn2+-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn2+-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn2+ into the intra-membrane Zn2+-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn2+ entry into the pore. This would allow dynamic coupling of Zn2+ transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn2+ levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.