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Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+/Cl− exchanger

Nature Structural & Molecular Biology volume 19, pages 525–531 (2012)Cite this article

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Abstract

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H+/Cl− exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a crucial glutamate. The simultaneous binding of H+ and Cl− gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl− transport pathway identically alter the stoichiometries of H+/Cl− exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.

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Figure 1: Alternating-access transport.
Figure 2: Synergistic binding of H+ and Cl− to WT CLC-ec1.
Figure 3: The E148A mutant uncouples Cl− and H+ binding.
Figure 4: Cl−-coupled H+ binding to the Y445L mutant.
Figure 5: Cl−-coupled H+ binding to the E203Q mutant.
Figure 6: Modulation of the free energy of protonation of Glu148 and Glu203 by Cl− occupancy of the anion transport pathway.
Figure 7: pH dependence of the Cl− half-cycle of CLC-ec1.

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Acknowledgements

The authors thank C. Miller (Brandeis University) for the gift of 36Cl− and D. Posson, D. Basilio, M. Malvezzi, C. Nimigean and H. Weinstein for helpful discussions and comments on the manuscript. This work was supported by grants from the National Institutes of Health (1R01GM085232 to A.A.) and by a grant from the Swiss National Science Foundation (SNF-Professorship #118928 to S.B.). Y.X. is supported by the China Scholarship Council. Part of the simulations were performed using the facilities of the Swiss National Supercomputing Centre (CSCS).

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Authors and Affiliations

  1. Department of Anesthesiology, Weill Cornell Medical College, New York, New York, USA

    Alessandra Picollo & Alessio Accardi

  2. Swiss Institute of Bioinformatics, Biozentrum, University of Basel, Basel, Switzerland

    Yanyan Xu, Niklaus Johner & Simon Bernèche

  3. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Niklaus Johner & Alessio Accardi

  4. Department of Biochemistry, Weill Cornell Medical College, New York, New York, USA

    Alessio Accardi

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  1. Alessandra Picollo
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Contributions

A.P. performed the ITC measurements; A.A. and A.P. performed the flux experiments and analyzed data; Y.X., N.J. and S.B. performed and analyzed the simulations; A.A. designed research and wrote the paper; all authors contributed to the editing of the manuscript.

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Correspondence to Simon Bernèche or Alessio Accardi.

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Picollo, A., Xu, Y., Johner, N. et al. Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+/Cl− exchanger. Nat Struct Mol Biol 19, 525–531 (2012). https://doi.org/10.1038/nsmb.2277

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