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Crystal structure of a eukaryotic phosphate transporter

Abstract

Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors1,2,3,4,5. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical ‘rocker-switch’ mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance6,7. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.

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Figure 1: Structure of the high-affinity phosphate transporter, PiPT.
Figure 2: The proposed proton exit pathway.
Figure 3: Proposed mechanism of phosphate transport.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank with the accession number 4J05.

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Acknowledgements

We thank K. Giacomini for discussions about SLC transporters; P. Nissen for comments that improved the manuscript; J. Holton, G. Meigs, C. Ogata and N. Venugopalan for assistance with synchrotron data collection at the Advanced Light Source and Advanced Photon Source; and C. Waddling, P. Wassam and M. Tessema for technical assistance. B.P.P. was supported by a postdoctoral fellowship from the Carlsberg Foundation and later by a fellowship from the Danish Cancer Society; H.K. by a fellowship from Woods Whelan foundation, Council of Scientific and Industrial Research, Government of India, and a travel grant from Jawaharlal Nehru University, New Delhi.; A.Sc. by NIH postdoctoral fellowship F32 GM088991; A.Sa. by NIH grants U54 GM094625 and U01 GM61390; A.K.J. by a Research Assistant Professorship to do work at UCSF from the American Society for Microbiology; and R.M.S. by NIH grants U54 GM094625, GM24485 and GM073210.

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Authors

Contributions

B.P.P. did expression, purification and crystallization experiments, collected and processed the data, and determined, refined and analysed the structure. H.K. identified the target, did purification and crystallization experiments, collected data and identified the use of NG for crystallization. A.B.W. optimized the yeast expression system, and assisted in data collection and data analysis. A.J.R. helped with protein purification and crystallization. Z.R.-Z. assisted in optimization of the yeast expression system, cloned, expressed, purified and characterized the target, and set up initial crystallization experiments. B.H.C. did cloning, expression tests and cell growth. A.Sc. performed bioinformatics analysis and built human homology models. M.B. did molecular dynamics and analysed the results. W.H. trained H.K. and assisted H.K. in data collection. A.Sa. supervised homology modelling, bioinformatics analysis and molecular dynamics. A.K.J. identified the target and initiated the project. R.M.S. supervised the project and analysed the structure. B.P.P. and R.M.S. wrote the paper with input from H.K., A.B.W., A.Sc., A.Sa. and A.K.J.

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Correspondence to Robert M. Stroud.

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The authors declare no competing financial interests.

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Pedersen, B., Kumar, H., Waight, A. et al. Crystal structure of a eukaryotic phosphate transporter. Nature 496, 533–536 (2013). https://doi.org/10.1038/nature12042

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