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Allosteric substrate switching in a voltage-sensing lipid phosphatase

Nature Chemical Biology volume 12, pages 261–267 (2016)Cite this article

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Abstract

Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

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Figure 1: Membrane-associated FRET reporters respond rapidly to changes in PIP2.
Figure 2: PIP3→PIP2 activity occurs soon after VSD rearrangement associated with gating-charge displacement.
Figure 3: Wild-type Ci-VSP appears to have two active enzymatic states.
Figure 4: VSD mutants stabilize discrete VSD conformations.
Figure 5: VSD mutants stabilize discrete enzyme activity states.
Figure 6: Two-step VSD conformational control over VSP phosphatase with two active states.

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Acknowledgements

We thank Y. Okamura (Osaka University) for Ci-VSP, M. Matsuda (Kyoto University) for Fllip-pm, T. Meyer (Stanford University) for GFP-PLC-PH, T. Balla (US National Institutes of Health) for GFP-TAPP-PH, H. Okada, C. Stanley and Z. Fu for technical support, as well as A. Reiner, S. Bharill, S. Kohout, E. Carroll and other current and former members of the Isacoff laboratory for guidance on analysis and helpful discussions. This work was supported by the US National Institutes of Health (R01GM117051 and T32GM008295; E.Y.I.) as well as fellowship support for provided by the UC Berkeley Chancellors Fellowship for Graduate Study (S.S.G.).

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Authors and Affiliations

  1. Biophysics Graduate Group, University of California, Berkeley, California, USA

    Sasha S Grimm & Ehud Y Isacoff

  2. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

    Ehud Y Isacoff

  3. Helen Wills Neuroscience Institute, University of California, Berkeley, California, USA

    Ehud Y Isacoff

  4. Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Ehud Y Isacoff

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  1. Sasha S Grimm
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E.Y.I. and S.S.G. conceived the study, analyzed the data, directed the evolution of the project and wrote the paper. S.S.G. conducted the experiments.

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Correspondence to Ehud Y Isacoff.

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Grimm, S., Isacoff, E. Allosteric substrate switching in a voltage-sensing lipid phosphatase. Nat Chem Biol 12, 261–267 (2016). https://doi.org/10.1038/nchembio.2022

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