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A water-mediated allosteric network governs activation of Aurora kinase A

Nature Chemical Biology volume 13, pages 402–408 (2017)Cite this article

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Abstract

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

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Figure 1: Tpx2 induces a population shift toward the DFG-in state.
Figure 2: Tpx2 promotes a nanometer-scale shift of the activation loop.
Figure 3: The Q185 spine residue participates in a water-mediated allosteric network.
Figure 4: The Q185 residue is essential for allosteric activation of AurA.
Figure 5: A PKA-like water network supports activation of AurA by Tpx2, but not by phosphorylation.

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Acknowledgements

We thank E. Conti (Max Planck Institute of Biochemistry, Martinsried) for providing the DNA construct encoding WT human AurA. We thank R. Frontiera for helpful discussions and advice on fitting infrared spectra, and J. Muretta for many fruitful discussions and advice on fluorescence spectroscopy. We thank T. Freedman and W. Gordon for helpful discussions and critical reading of the manuscript. This work was funded in part by grants from the National Institutes of Health (R00 GM102288, N.M.L.). J.D.C. acknowledges support from the Sloan Kettering Institute and NIH grant P30 CA008748. The authors are grateful to the Folding@home donors who participated in this project.

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Author notes

  1. Soreen Cyphers and Emily F Ruff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, University of Minnesota, Twin Cities, Minneapolis, Minnesota, USA

    Soreen Cyphers, Emily F Ruff & Nicholas M Levinson

  2. Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Julie M Behr & John D Chodera

  3. Tri-Institutional Program in Computational Biology and Medicine, Weill Cornell Medical College, New York, New York, USA

    Julie M Behr

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  1. Soreen Cyphers
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Contributions

N.M.L. conceived and designed the project. S.C. performed the kinase activity assays and thermal shift assays, and S.C. and N.M.L. performed the infrared spectroscopy experiments. E.F.R. performed the fluorescence experiments. J.D.C., and J.M.B. conceived and performed the molecular dynamics simulations and analysis. N.M.L. and J.D.C. wrote the manuscript.

Corresponding author

Correspondence to Nicholas M Levinson.

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J.D.C. is a member of the Scientific Advisory Board for Schrödinger, LLC.

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Cyphers, S., Ruff, E., Behr, J. et al. A water-mediated allosteric network governs activation of Aurora kinase A. Nat Chem Biol 13, 402–408 (2017). https://doi.org/10.1038/nchembio.2296

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