Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of human PLD1 provides insight into activation by PI(4,5)P2 and RhoA

Nature Chemical Biology volume 16, pages 400–407 (2020)Cite this article

Subjects

Abstract

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structure of human PLD1.
Fig. 2: Structural comparison of PLD family phosphodiesterases with human PLD1.
Fig. 3: Active-site cavity in PLD1.
Fig. 4: A phosphohistidine intermediate helps define the catalytic cycle of PLD1.
Fig. 5: A polybasic pocket defines a new PIP2 activation site at the membrane.
Fig. 6: Model of RhoA activation of PLD1.

Similar content being viewed by others

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 6U8Z. Source data for Figs. 1 and 3–5 are presented with the paper.

References

  1. Bruntz, R. C., Lindsley, C. W. & Brown, H. A. Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer. Pharm. Rev. 66, 1033–1079 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jenkins, G. M. & Frohman, M. A. Phospholipase D: a lipid centric review. Cell Mol. Life Sci. 62, 2305–2316 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Tanguy, E., Wang, Q. & Vitale, N. Role of phospholipase D-derived phosphatidic acid in regulated exocytosis and neurological disease. In Handbook of Experimental Pharmacology 1–16 (Springer, 2018).

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

    Article  CAS  PubMed  Google Scholar 

  5. Chen, Q. et al. Key roles for the lipid signaling enzyme phospholipase D1 in the tumor microenvironment during tumor angiogenesis and metastasis. Sci. Signal. 5, ra79 (2012).

    PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Göbel, K. et al. Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis. Eur. J. Immunol. 44, 2295–2305 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Kang, D. W. et al. Phospholipase D1 has a pivotal role in interleukin-1β-driven chronic autoimmune arthritis through regulation of NF-κB, hypoxia-inducible factor 1α and foxo3a. Mol. Cell. Biol. 33, 2760–2772 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lindsley, C. W. & Brown, H. A. Phospholipase D as a therapeutic target in brain disorders. Neuropsychopharmacology 37, 301–302 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Elvers, M. et al. Impaired αIIbβ3 integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1. Sci. Signal. 3, ra1 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  13. Su, W. et al. 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase D pharmacological inhibitor that alters cell spreading and inhibits chemotaxis. Mol. Pharmacol. 75, 437–446 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Stegner, D. et al. Pharmacological inhibition of phospholipase D protects mice from occlusive thrombus formation and ischemic stroke. Arterioscler. Thromb. Vasc. Biol. 33, 2212–2217 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. 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).

    Article  CAS  PubMed  Google Scholar 

  16. Colley, W. C. et al. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr. Biol. 7, 191–201 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Hammond, S. M. et al. Characterization of two alternately spliced forms of phospholipase D1. J. Biol. Chem. 272, 3860–3868 (1997).

    Article  CAS  PubMed  Google Scholar 

  20. Yamazaki, M. et al. Interaction of the small G protein RhoA with the C terminus of human phospholipase D1. J. Biol. Chem. 274, 6035–6038 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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).

    Article  CAS  PubMed  Google Scholar 

  24. Leiros, I., Secundo, F., Zambonelli, C., Servi, S. & Hough, E. The first crystal structure of a phospholipase D. Structure 8, 655–667 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Cai, S. & Exton, J. H. Determination of interaction sites of phospholipase D1 for RhoA. Biochem J. 355, 779–785 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Du, G. et al. Dual requirement for Rho and protein kinase C in direct activation of phospholipase D1 through G protein-coupled receptor signaling. Mol. Biol. Cell 11, 4359–4368 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang, Y., Altshuller, Y. M., Hammond, S. M., Morris, A. J. & Frohman, M. A. Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C. EMBO J. 18, 6339–6348 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gottlin, E. B., Rudolph, A. E., Zhao, Y., Matthews, H. R. & Dixon, J. E. Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. Proc. Natl Acad. Sci. USA 95, 9202–9207 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Pierce, B. G. et al. ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 30, 1771–1773 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Henage, L. G., Exton, J. H. & Brown, H. A. Kinetic analysis of a mammalian phospholipase D. J. Biol. Chem. 281, 3408–3417 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Hicks, S. N. et al. General and versatile autoinhibition of PLC isozymes. Mol. Cell 31, 383–394 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lyon, A. M. et al. An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation. Nat. Struct. Mol. Biol. 18, 999–1005 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lyon, A. M., Begley, J. A., Manett, T. D. & Tesmer, J. J. Molecular mechanisms of phospholipase C β3 autoinhibition. Structure 22, 1844–1854 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Henage, L. G. Kinetic analysis of a mammalian phospholipase D. J. Biol. Chem. 281, 3408–3417 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Ali, I. et al. Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae. Acta Crystallogr. F 74, 315–321 (2018).

    Article  CAS  Google Scholar 

  39. Cronin, C. N., L, K. B. & Rogers, Joe. Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Sci. 16, 2023–2029 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Waterman, D. G. et al. Diffraction-geometry refinement in the DIALS framework. Acta Cryst. 72, 558–575 (2016).

    CAS  Google Scholar 

  41. Pothineni, S. B. et al. Tightly integrated single- and multi-crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system. J. Appl. Cryst. 47, 1992–1999 (2014).

    Article  CAS  Google Scholar 

  42. Terwilliger, T. C. et al. Can I solve my structure by SAD phasing? Planning an experiment, scaling data and evaluating the useful anomalous correlation and anomalous signal. Acta Crystallogr. D 72, 359–374 (2016).

    Article  CAS  Google Scholar 

  43. Grosse-Kunstleve, R. W. & Adams, P. D. Substructure search procedures for macromolecular structures. Acta Crystallogr. D 59, 1966–1973 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582–601 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuildwizard. Acta Crystallogr. D 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Morris, A. J., Frohman, M. A. & Engebrecht, J. Measurement of phospholipase D activity. Anal. Biochem. 252, 1–9 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Philip, F., Ha, E. E., Seeliger, M. A. & Frohman, M. A. Measuring phospholipase D enzymatic activity through biochemical and imaging methods. Methods Enzymol. 583, 309–325 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the staff at the FMX and GM/CA-CAT beamlines for assistance during data collection. Beamline FMX (17-ID-2) is operated by LSBR, supported by NIH/NIGMS (P41GM111244) and DOE/BER (KP1605010). GM/CA@APS has been funded in whole or in part with federal funds from the NCI (ACB-12002) and the NIGMS (AGM-12006). The Eiger 16M detector was funded by an NIH–Office of Research Infrastructure Programs High-End Instrumentation Grant (S10 OD012289). This research used resources of the APS, a US Department of Energy (DOE) Office of Science User Facilities operated for the DOE Office of Science by Argonne National Laboratory (under contract no. DE-AC02-06CH11357). This work was also supported by NIH awards R35GM128666 (M.V.A.), T32GM092714 (F.Z.B.) and R01GM084251 (M.A.F.), NSF award 1612689 (C.M.S.), a Carol Baldwin Breast Cancer Award (M.A.F.) and a Chhabra-URECA award (J.A.B.).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA

    Forrest Z. Bowling, Justin A. Bell, Tahrima S. Huq & Michael V. Airola

  2. Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA

    Christian M. Salazar & Michael A. Frohman

Authors
  1. Forrest Z. Bowling
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian M. Salazar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Justin A. Bell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tahrima S. Huq
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael A. Frohman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael V. Airola
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.Z.B. performed all protein purifications, crystallization experiments, liposome sedimentation assays, docking experiments and in vitro activity assays. C.M.S. performed all cell-based activity assays. J.A.B. and T.S.H. constructed key plasmids. F.Z.B. and M.V.A. determined and refined the final crystal structure. F.Z.B., M.A.F. and M.V.A. contributed intellectual and strategic input. M.A.F. and M.V.A. supervised work. F.Z.B., M.A.F. and M.V.A. wrote and edited the final manuscript. All authors approved the final manuscript.

Corresponding author

Correspondence to Michael V. Airola.

Ethics declarations

Competing interests

The authors declare no competing interests.

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 Table 1 and Figs. 1 and 2.

Reporting Summary

RhoA and lipid modeled structures

Structural CIF files

Source data

Source Data

Source data for figs. 1 and 3–5

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bowling, F.Z., Salazar, C.M., Bell, J.A. et al. Crystal structure of human PLD1 provides insight into activation by PI(4,5)P2 and RhoA. Nat Chem Biol 16, 400–407 (2020). https://doi.org/10.1038/s41589-020-0499-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0499-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing