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A glutamate switch controls voltage-sensitive phosphatase function

Nature Structural & Molecular Biology volume 19, pages 633–641 (2012)Cite this article

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Abstract

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) couples a voltage-sensing domain (VSD) to a lipid phosphatase that is similar to the tumor suppressor PTEN. How the VSD controls enzyme function has been unclear. Here, we present high-resolution crystal structures of the Ci-VSP enzymatic domain that reveal conformational changes in a crucial loop, termed the 'gating loop', that controls access to the active site by a mechanism in which residue Glu411 directly competes with substrate. Structure-based mutations that restrict gating loop conformation impair catalytic function and demonstrate that Glu411 also contributes to substrate selectivity. Structure-guided mutations further define an interaction between the gating loop and linker that connects the phosphatase to the VSD for voltage control of enzyme activity. Together, the data suggest that functional coupling between the gating loop and the linker forms the heart of the regulatory mechanism that controls voltage-dependent enzyme activation.

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Figure 1: Structure of the Ci-VSP catalytic domain and comparison with PTEN.
Figure 2: Comparison of Ci-VSP intracellular domain structures reveals gating loop movements.
Figure 3: Comparison of Ci-VSP active site P-loop conformations.
Figure 4: Functional studies of Ci-VSP active site mutants.
Figure 5: Tests of gating loop functional importance.
Figure 6: Structure of a Ci-VSP–IP3 complex.
Figure 7: Ci-VSP linker–gating loop coupling.

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Acknowledgements

This work was supported by grants to D.L.M. from the US National Institutes of Health (NIH), R01 DC007664, and the American Heart Association (AHA), 0740019N, and to E.Y.I. from the NIH, R01 NS035549 and U24 NS057631. Ci-VSP in the pSD64TF vector was provided by Y. Okamura (Osaka University). GFP-PLC-PH, GFP-TAPP-PH and GFP-GRP-PH were provided by T. Meyer (Stanford University), T. Balla (NIH) and J. Falke (University of Colorado), respectively. Kir2.1 was provided by E. Reuveny (Weizmann Institute of Science). We thank members of the Minor and Isacoff labs for support throughout these studies. D.L.M. is an AHA Established Investigator.

Author information

Author notes

  1. Qiang Xu

    Present address: Present address: Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.,

  2. Lijun Liu and Susy C Kohout: These authors contributed equally to this work.

Authors and Affiliations

  1. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA

    Lijun Liu, Qiang Xu, Simone Müller, Christopher R Kimberlin & Daniel L Minor Jr

  2. California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, California, USA.,

    Lijun Liu, Qiang Xu, Simone Müller, Christopher R Kimberlin & Daniel L Minor Jr

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

    Susy C Kohout & Ehud Y Isacoff

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

    Ehud Y Isacoff & Daniel L Minor Jr

  5. Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Ehud Y Isacoff

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

    Ehud Y Isacoff

  7. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA

    Daniel L Minor Jr

  8. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.,

    Daniel L Minor Jr

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Contributions

L.L., S.C.K., S.M., E.Y.I. and D.L.M. conceived the study. L.L., S.C.K., Q.X., S.M. and C.R.K. conducted the experiments and analyzed data. E.Y.I. and D.L.M. analyzed data and provided guidance and support throughout. L.L., S.C.K., E.Y.I. and D.L.M. wrote the paper.

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Correspondence to Daniel L Minor Jr.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–4 (PDF 3378 kb)

Supplementary Movie 1

Morph of 241 Form I copy B (open) and 241 Form II copy B (closed) structures. Members of the hydrophobic α456 pocket are highlighted in yellow. Other colors are as in Figure 1. (MP4 28767 kb)

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Liu, L., Kohout, S., Xu, Q. et al. A glutamate switch controls voltage-sensitive phosphatase function. Nat Struct Mol Biol 19, 633–641 (2012). https://doi.org/10.1038/nsmb.2289

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