Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Miki Kawachi, Yoshihiro Kobae, Sayaka Kogawa, Tetsuro Mimura, Ute Krämer, Masayoshi Maeshima

研究成果: Article同行評審

36 引文 斯高帕斯(Scopus)

摘要

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn 2+ ?H + antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn 2+ 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 Zn 2+-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 Zn 2+-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 Zn 2+ into the intra-membrane Zn 2+-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn 2+ entry into the pore. This would allow dynamic coupling of Zn 2+ transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn 2+ 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.

原文English
頁(從 - 到)2339-2356
頁數18
期刊FEBS Journal
279
發行號13
DOIs
出版狀態Published - 2012 7月

All Science Journal Classification (ASJC) codes

  • 生物化學
  • 分子生物學
  • 細胞生物學

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