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Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity

Nature Plants volume 3, pages 825–832 (2017)Cite this article

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Abstract

The triose-phosphate/phosphate translocator (TPT) catalyses the strict 1:1 exchange of triose-phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous study for more than 40 years, the molecular mechanism of TPT is poorly understood because of the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocker-switch motion of helix bundles, and molecular dynamics simulations support a model in which this rocker-switch motion is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT.

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Fig. 1: Functional characterization of GsGPT.
Fig. 2: Overall structure and substrate recognition of GsGPT.
Fig. 3: Comparison of the pPT subtypes.
Fig. 4: Occluded structure and alternating-access.
Fig. 5: Molecular dynamics simulations.
Fig. 6: Proposed model for the strict 1:1 exchange.

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Acknowledgements

We thank H. Nishimasu for comments on the manuscript; A. Kurabayashi, K. Ogomori, W. Shihoya and R. Taniguchi for technical assistance; and the beam-line scientists at SPring-8 BL32XU for assistance in data collection. The diffraction experiments were performed at SPring-8 BL32XU (proposals 2015B0119, 2015B2057 and 2016B2527). Computations of MD simulations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. This work was supported by grants from the Platform for Drug Discovery, Informatics and Structural Life Science by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), JSPS KAKENHI (Grant Nos. 24227004, 25291011), the FIRST program, JST PRESTO; and a Grant-in-Aid for JSPS Fellows (Grant No. 16J07405).

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Authors and Affiliations

  1. Department of Biological Sciences, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan

    Yongchan Lee, Tomohiro Nishizawa, Mizuki Takemoto, Kaoru Kumazaki, Ryuichiro Ishitani & Osamu Nureki

  2. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan

    Tomohiro Nishizawa & Kunio Hirata

  3. RIKEN SPring-8 Center, Hyogo, 679-5148, Japan

    Keitaro Yamashita & Kunio Hirata

  4. Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan

    Ayumi Minoda

  5. Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

    Satoru Nagatoishi & Kouhei Tsumoto

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  1. Yongchan Lee
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Contributions

Y.L. designed the research, expressed, purified and crystallized GsGPT, determined the structures and performed biochemical assays. Y.L., K.Y. and K.H. collected and processed diffraction data. T.N. and K.K. assisted with the structure determination. M.T. performed the molecular dynamics simulations. A.M. prepared G. sulphuraria cDNA. S.N. and K.T. performed the SEC-MALLS experiment. Y.L., T.N., R.I. and O.N. wrote the manuscript with help from all authors. O.N. directed and supervised all of the research.

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Correspondence to Osamu Nureki.

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Supplementary Information

Supplementary Figures 1–11, Supplementary Tables 1 and 2

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Supplementary Data 1

Multiple sequence alignment between human NSTs, Arabidopsis NST/TPT members and GsGPT

Supplementary Video 1

Conformational change of GsGPT

Supplementary Video 2

Molecular dynamics simulation of GsGPT without Pi

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Lee, Y., Nishizawa, T., Takemoto, M. et al. Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity. Nature Plants 3, 825–832 (2017). https://doi.org/10.1038/s41477-017-0022-8

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