Abstract
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor–hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes.
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Data availability
Structure data that support the findings of this study have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under the following accession codes: PLD2-WO4, 6OHM; PLD2-apo, 6OHO; PLD2-1, 6OHP; PLD2-3, 6OHS; PLD2-4, 6OHQ; PLD1-5, 6OHR. The data that support the remaining findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We acknowledge the Paul Scherrer Institut, Villigen, Switzerland for provision of synchrotron radiation beamtime at beamlines PXI and PXII of the SLS and thank J. Diez at Expose GMBH for data collection at SLS and D. Esposito at Leidos (Frederick National Laboratory) for T. ni cells and small-scale expression and purification of hPLD1b.
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J.V.C. solved the structures of hPLD2 and C.M.M. solved the structure of hPLD1b. M.C. and P.R.K. expressed and purified hPLD proteins. I.J.E. performed modeling experiments and K.M. performed SPR experiments. E.A.P., T.L.M.-D. and T.C. contributed to compound design and synthesis. J.C.S., P.M. and K.A.S. performed biochemical assays. All authors contributed to data interpretation. J.V.C., C.M.M. and E.A.P. wrote the manuscript.
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Biogen funded these studies. C.M.M., E.A.P., J.C.S., I.J.E., P.M., T.C., K.M., M.C., K.A.S., P.R.K., T.L.M.-D. and J.V.C. are employees and shareholders of Biogen.
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Metrick, C.M., Peterson, E.A., Santoro, J.C. et al. Human PLD structures enable drug design and characterization of isoenzyme selectivity. Nat Chem Biol 16, 391–399 (2020). https://doi.org/10.1038/s41589-019-0458-4
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DOI: https://doi.org/10.1038/s41589-019-0458-4
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