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Crystal structure of plant vacuolar iron transporter VIT1

Abstract

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.

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Data availability

Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. Coordinates and structure factors have been deposited in the Protein Data Bank, with the accession codes PDB 6IU3 and 6IU4 for non-soaked and Co2+-soaked EgVIT123-249, and 6IU5, 6IU6, 6IU8, and 6IU9 for Zn2+-bound, Ni2+-soaked, Co2+-soaked, and Fe2+-soaked EgVIT170-144, respectively. X-ray diffraction images are also available at Zenodo data repository (https://zenodo.org/record/2532136 for full length with Co/Zn; https://zenodo.org/record/2532134 for MBD; https://zenodo.org/record/2532138 for SIR datasets).

Additional information

Journal peer review information Nature Plants thanks Rachelle Gaudet, Simon Newstead and other anonymous reviewers for their contribution to the peer review of this work

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank all of the members of the Nureki laboratory, especially H. Nishimasu, H. Hirano, S. Hirano, and W. Shihoya for data collection at SLS and SPring-8; A. Kurabayashi and M. Miyazaki for technical assistance in the research; and the beamline staff members at PXI X06SA of the Swiss Light Source (Villigen, Switzerland), 05A of the Taiwan Photon Source (Hsinchu, Taiwan) and BL32XU of SPring-8 (Hyogo, Japan). The diffraction experiments were performed at the Swiss Light Source and at SPring-8 BL32XU and BL41XU (proposals 2015A1024, 2015A1057, 2015B2024, 2015B2057, and 2016A2527), with the approval of RIKEN. This work was supported by a MEXT Grant-in-Aid for Specially Promoted Research (grant 16H06294) to O.N. This work was also supported by JSPS KAKENHI (grant 17J06203 to T.K.; 15H06862 to K.Y.; 17H05000 to T. Nishizawa; 16H06574 to R.I.). H.K. and T. Nishizawa. were funded by JST, PRESTO (grants JPMJPR14L9 and JPMJPR14L8, respectively).

Author information

T.K. purified and crystallized EgVIT1, determined the structure, planned the mutational analyses, and performed the liposome assays, under the supervision of K.K., R.T., R.I., T. Nishizawa, and O.N. K.Y. and K.H. assisted with the diffraction data collection and the data analyses. M.W. performed the yeast spot analysis and western blotting, under the supervision of K.I. T. Nakane and T. Nishizawa assisted with the data analyses. R.I., T.K., T. Nishizawa, and O.N. wrote the manuscript, with feedback from all of the authors. T. Nishizawa and O.N. supervised the research.

Competing interests

The authors declare no competing interests.

Correspondence to Tomohiro Nishizawa or Osamu Nureki.

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Supplementary Figures 1–10, and Supplementary Tables 1 and 2.

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Fig. 1: Functional analysis of EgVIT1.
Fig. 2: Overall structure of EgVIT1.
Fig. 3: Hydrophilic pocket of the TMD.
Fig. 4: Structure and functional analysis of the MBD.
Fig. 5: Co2+ binding within the cytoplasmic pocket.
Fig. 6: Proposed model for metal ion transport by EgVIT1.