Skip to main content

Advertisement

SpringerLink
Log in

Human PLD structures enable drug design and characterization of isoenzyme selectivity

  • Article
  • Published:

From Nature Chemical Biology

View current issue Submit your manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1: The catalytic domain of hPLD2 adopts the conserved PLD fold with additional structural features.
Fig. 2: Biophysical and structural characterization of the catalytic pockets of PLDs.
Fig. 3: The F614 pocket is important for achieving hPLD inhibitor selectivity.
Fig. 4: The novel cavity in the hPLD2 catalytic pocket inspired the design of an inhibitor that enabled hPLD1b crystallization.
Fig. 5: Mutation analysis of the catalytic pocket identifies residues involved in isoenzyme selectivity.

Similar content being viewed by others

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.

References

  1. Peng, X. & Frohman, M. A. Mammalian phospholipase D physiological and pathological roles. Acta Physiol. (Oxf.) 204, 219–226 (2012).

    CAS  Google Scholar 

  2. Arhab, Y., Abousalham, A. & Noiriel, A. Plant phospholipase D mining unravels new conserved residues important for catalytic activity. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1864, 688–703 (2019).

    CAS  PubMed  Google Scholar 

  3. Celma, L. et al. Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity. PLoS One 12, e0189049 (2017).

    PubMed  PubMed Central  Google Scholar 

  4. Bozatzi, P. & Sapkota, G. P. The FAM83 family of proteins: from pseudo-PLDs to anchors for CK1 isoforms. Biochem. Soc. Trans. 46, 761–771 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sung, T. C. et al. Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 16, 4519–4530 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Secundo, F., Carrea, G., D’Arrigo, P. & Servi, S. Evidence for an essential lysyl residue in phospholipase D from Streptomyces sp. by modification with diethyl pyrocarbonate and pyridoxal 5-phosphate. Biochemistry 35, 9631–9636 (1996).

    CAS  PubMed  Google Scholar 

  7. Iwasaki, Y., Horiike, S., Matsushima, K. & Yamane, T. Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain. Eur. J. Biochem. 264, 577–581 (1999).

    CAS  PubMed  Google Scholar 

  8. Ponting, C. P. & Kerr, I. D. A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. Protein Sci. 5, 914–922 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Koonin, E. V. A duplicated catalytic motif in a new superfamily of phosphohydrolases and phospholipid synthases that includes poxvirus envelope proteins. Trends Biochem. Sci. 21, 242–243 (1996).

    CAS  PubMed  Google Scholar 

  10. Sung, T. C., Altshuller, Y. M., Morris, A. J. & Frohman, M. A. Molecular analysis of mammalian phospholipase D2. J. Biol. Chem. 274, 494–502 (1999).

    CAS  PubMed  Google Scholar 

  11. Selvy, P. E., Lavieri, R. R., Lindsley, C. W., & Brown, H. A. Phospholipase D: enzymology, functionality, and chemical modulation. Chem. Rev. 111, 6064–6119 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Leiros, I., McSweeney, S. & Hough, E. The reaction mechanism of phospholipase D from Streptomyces sp. strain PMF. Snapshots along the reaction pathway reveal a pentacoordinate reaction intermediate and an unexpected final product. J. Mol. Biol. 339, 805–820 (2004).

    CAS  PubMed  Google Scholar 

  13. Leiros, I. et al. Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces sp. Acta Crystallogr. D 56, 466–468 (2000).

    CAS  PubMed  Google Scholar 

  14. Uesugi, Y. & Hatanaka, T. Phospholipase D mechanism using Streptomyces PLD. Biochim. Biophys. Acta 1791, 962–969 (2009).

    CAS  PubMed  Google Scholar 

  15. Hammond, S. M. et al. Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J. Biol. Chem. 272, 3860–3868 (1997).

    CAS  PubMed  Google Scholar 

  16. Colley, W. C. et al. Cloning and expression analysis of murine phospholipase D1. Biochem J. 326, 745–753 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Nelson, R. K. & Frohman, M. A. Physiological and pathophysiological roles for phospholipase D. J. Lipid Res. 56, 2229–2237 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Frohman, M. A. The phospholipase D superfamily as therapeutic targets. Trends Pharm. Sci. 36, 137–144 (2015).

    CAS  PubMed  Google Scholar 

  19. Gomez-Cambronero, J. & Ganesan, R. Targeting phospholipase D genetically and pharmacologically for studying leukocyte function. Methods Mol. Biol. 1835, 297–314 (2018).

    CAS  PubMed  Google Scholar 

  20. Brown, H. A., Thomas, P. G. & Lindsley, C. W. Targeting phospholipase D in cancer, infection and neurodegenerative disorders. Nat. Rev. Drug Discov. 16, 351–367 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Waterson, A. G. et al. Isoform selective PLD inhibition by novel, chiral 2,8-diazaspiro[4.5]decan-1-one derivatives. Bioorg. Med. Chem. Lett. 28, 3670–3673 (2018).

    CAS  PubMed  Google Scholar 

  22. Aloulou, A., Rahier, R., Arhab, Y., Noiriel, A. & Abousalham, A. Phospholipases: an overview. Methods Mol. Biol. 1835, 69–105 (2018).

    CAS  PubMed  Google Scholar 

  23. Uesugi, Y., Arima, J., Iwabuchi, M. & Hatanaka, T. C-terminal loop of Streptomyces phospholipase D has multiple functional roles. Protein Sci. 16, 197–207 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Sung, T. C., Zhang, Y., Morris, A. J. & Frohman, M. A. Structural analysis of human phospholipase D1. J. Biol. Chem. 274, 3659–3666 (1999).

    CAS  PubMed  Google Scholar 

  25. Stuckey, J. A. & Dixon, J. E. Crystal structure of a phospholipase D family member. Nat. Struct. Biol. 6, 278–284 (1999).

    CAS  PubMed  Google Scholar 

  26. Morris, A. J., Engebrecht, J. & Frohman, M. A. Structure and regulation of phospholipase D. Trends Pharm. Sci. 17, 182–185 (1996).

    CAS  PubMed  Google Scholar 

  27. Hammond, S. M. et al. Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. J. Biol. Chem. 270, 29640–29643 (1995).

    CAS  PubMed  Google Scholar 

  28. Exton, J. H. Phospholipase D. Ann. N. Y. Acad. Sci. 905, 61–68 (2000).

    CAS  PubMed  Google Scholar 

  29. Liu, M. Y., Gutowski, S. & Sternweis, P. C. The C terminus of mammalian phospholipase D is required for catalytic activity. J. Biol. Chem. 276, 5556–5562 (2001).

    CAS  PubMed  Google Scholar 

  30. Sciorra, V. A. et al. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes. EMBO J. 18, 5911–5921 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Du, G. et al. Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs. J. Cell Biol. 162, 305–315 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. De Cuyper, H., van Praag, H. M. & Verstraeten, D. The clinical significance of halopemide, a dopamine-blocker related to the butyrophenones. Neuropsychobiology 12, 211–216 (1984).

    PubMed  Google Scholar 

  33. Lavieri, R. R. et al. Design, synthesis, and biological evaluation of halogenated N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)benzamides: discovery of an isoform-selective small molecule phospholipase D2 inhibitor. J. Med. Chem. 53, 6706–6719 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Lewis, J. A. et al. Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part I: impact of alternative halogenated privileged structures for PLD1 specificity. Bioorg. Med. Chem. Lett. 19, 1916–1920 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Monovich, L. et al. Optimization of halopemide for phospholipase D2 inhibition. Bioorg. Med. Chem. Lett. 17, 2310–2311 (2007).

    CAS  PubMed  Google Scholar 

  36. O’Reilly, M. C. et al. Development of dual PLD1/2 and PLD2 selective inhibitors from a common 1,3,8-triazaspiro[4.5]decane core: discovery of Ml298 and Ml299 that decrease invasive migration in U87-MG glioblastoma cells. J. Med. Chem. 56, 2695–2699 (2013).

    PubMed  PubMed Central  Google Scholar 

  37. Scott, S. A. et al. Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat. Chem. Biol. 5, 108–117 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Scott, S. A. et al. Discovery of desketoraloxifene analogues as inhibitors of mammalian, Pseudomonas aeruginosa, and NAPE phospholipase D enzymes. ACS Chem. Biol. 10, 421–432 (2015).

    CAS  PubMed  Google Scholar 

  39. Ganesan, R., Mahankali, M., Alter, G. & Gomez-Cambronero, J. Two sites of action for PLD2 inhibitors: the enzyme catalytic center and an allosteric, phosphoinositide biding pocket. Biochim. Biophys. Acta 1851, 261–272 (2015).

    CAS  PubMed  Google Scholar 

  40. Kam, Y. & Exton, J. H. Dimerization of phospholipase D isozymes. Biochem. Biophys. Res. Commun. 290, 375–380 (2002).

    CAS  PubMed  Google Scholar 

  41. O’Reilly, M. C. et al. Discovery of a highly selective PLD2 inhibitor (ML395): a new probe with improved physiochemical properties and broad-spectrum antiviral activity against influenza strains. ChemMedChem 9, 2633–2637 (2014).

    PubMed  PubMed Central  Google Scholar 

  42. O’Reilly, M. C., Scott, S. A., Brown, H. A. & Lindsley, C. W. Further evaluation of novel structural modifications to scaffolds that engender PLD isoform selective inhibition. Bioorg. Med. Chem. Lett. 24, 5553–5557 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. Abergel, C., Walburger, A., Chenivesse, S. & Lazdunski, C. Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli. Acta Crystallogr. D 57, 317–319 (2001).

    CAS  PubMed  Google Scholar 

  44. Mahankali, M., Alter, G. & Gomez-Cambronero, J. Mechanism of enzymatic reaction and protein–protein interactions of PLD from a 3D structural model. Cell Signal 27, 69–81 (2015).

    CAS  PubMed  Google Scholar 

  45. Frohman, M. A., Sung, T. C. & Morris, A. J. Mammalian phospholipase D structure and regulation. Biochim. Biophys. Acta 1439, 175–186 (1999).

    CAS  PubMed  Google Scholar 

  46. Panda, A. et al. Functional analysis of mammalian phospholipase D enzymes. Biosci. Rep. 38, BSR20181690 (2018).

    PubMed  PubMed Central  Google Scholar 

  47. Auerbach, A. Activation of endplate nicotinic acetylcholine receptors by agonists. Biochem. Pharm. 97, 601–608 (2015).

    CAS  PubMed  Google Scholar 

  48. Dvir, H., Silman, I., Harel, M., Rosenberry, T. L. & Sussman, J. L. Acetylcholinesterase: from 3D structure to function. Chem. Biol. Interact. 187, 10–22 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Pettitt, T. R., McDermott, M., Saqib, K. M., Shimwell, N. & Wakelam, M. J. Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells. Biochem. J. 360, 707–715 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Lerchner, A., Mansfeld, J., Kuppe, K. & Ulbrich-Hofmann, R. Probing conserved amino acids in phospholipase D (Brassica oleracea var. capitata) for their importance in hydrolysis and transphosphatidylation activity. Protein Eng. Des. Sel. 19, 443–452 (2006).

    CAS  PubMed  Google Scholar 

  51. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Google Scholar 

  54. Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. Saerens, D. et al. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J. Mol. Biol. 352, 597–607 (2005).

    CAS  PubMed  Google Scholar 

  59. Hall, D. R. & Enyedy, I. J. Computational solvent mapping in structure-based drug design. Future Med. Chem. 7, 337–353 (2015).

    CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Physical Biochemistry, Biotherapeutic and Medicinal Sciences, Biogen, Cambridge, MA, USA

    Claire M. Metrick, Klaus Michelsen, Michael Cullivan, Kerri A. Spilker, P. Rajesh Kumar & Jayanth V. Chodaparambil

  2. Biogen Postdoctoral Scientist Program, Biogen, Cambridge, MA, USA

    Claire M. Metrick

  3. Medicinal Chemistry, Biotherapeutic and Medicinal Sciences, Biogen, Cambridge, MA, USA

    Emily A. Peterson, Istvan J. Enyedy, TeYu Chen & Tricia L. May-Dracka

  4. Bioassays and High Throughput Screens, Biotherapeutic and Medicinal Sciences, Biogen, Cambridge, MA, USA

    Joseph C. Santoro & Paramasivam Murugan

Authors
  1. Claire M. Metrick
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emily A. Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph C. Santoro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Istvan J. Enyedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paramasivam Murugan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. TeYu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Klaus Michelsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael Cullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kerri A. Spilker
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. P. Rajesh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Tricia L. May-Dracka
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jayanth V. Chodaparambil
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Jayanth V. Chodaparambil.

Ethics declarations

Competing interests

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–4 and Note.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0458-4

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation