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The molecular basis of phosphate discrimination in arsenate-rich environments

Nature volume 491, pages 134–137 (2012)Cite this article

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Abstract

Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pKa values1,2, similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches4,5. However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter’s presence therefore decouples metabolism6,7. Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-ångström-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole–anion interactions8,9, and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 103 excess) even in highly arsenate-rich environments.

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Figure 1: The phosphate–arsenate selectivity of PBPs.
Figure 2: Binding of arsenate and phosphate to PfluDING.
Figure 3: The (−)CAHB angles are optimal in the phosphate-bound structure but distorted with arsenate.

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Accession codes

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Protein Data Bank

Data deposits

Coordinates and structure factors for the arsenate-bound PfluDING structures at pH4.5 and 8.5 are deposited in the Protein Data Bank (accession codes 4F18 and 4F19); the phosphate-bound PfluDING coordinates and structures at pH4.5 and 8.5 (re-refinement) are deposited in the Protein Data Bank (accessioncodes 4F1U and 4F1V).

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Acknowledgements

We thank G. Gotthard for valuable inputs, C. Jelsch for help with the software Mopro, and A. Toth-Petroczy for help with the phylogenetic analysis. Financial support by the Israel Science Foundation is gratefully acknowledged. M.E. is a fellow supported by the Intra-European Fellowships Marie Curie program (grant No. 252836). D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry. T.J.E. was supported by an Eidgenössische Technische Hochschule fellowship.

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Author notes

  1. Mikael Elias and Alon Wellner: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

    Mikael Elias, Alon Wellner, Korina Goldin-Azulay & Dan S. Tawfik

  2. Unité de recherche sur les maladies infectieuses et tropicales émergentes, Faculté de Médecine et de Pharmacie, CNRS-Université de la Méditerranée, 13385 Marseille, France,

    Eric Chabriere

  3. Institute of Microbiology, Eidgenössische Technische Hochschule Zurich, Wolfgang Pauli Strasse 10, 8093 Zurich, Switzerland,

    Julia A. Vorholt & Tobias J. Erb

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Contributions

M.E. initiated the project, designed experiments, crystallized the proteins, collected and processed crystallographic data, performed discrimination assays, analysed the data and wrote the manuscript. A.W. cloned, purified and crystallized proteins, performed discrimination assays and analysed data. K.G.A. cloned and constructed the mutants, purified proteins and performed discrimination assays. T.J.E. and J.A.V. designed, performed and analysed reverse transcription PCR, as well as cellular experiments, and provided physiological data. E.C. purified proteins and collected diffraction data. D.S.T. initiated the project, designed experiments, analysed data and wrote the manuscript.

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Correspondence to Mikael Elias or Dan S. Tawfik.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-13, Supplementary Methods, Supplementary Tables 1-18 and additional references. (PDF 3199 kb)

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Elias, M., Wellner, A., Goldin-Azulay, K. et al. The molecular basis of phosphate discrimination in arsenate-rich environments. Nature 491, 134–137 (2012). https://doi.org/10.1038/nature11517

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