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. Map** 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.
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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).
Jenkins, G. M. & Frohman, M. A. Phospholipase D: a lipid centric review. Cell Mol. Life Sci. 62, 2305–2316 (2005).
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).
Frohman, M. A. The phospholipase D superfamily as therapeutic targets. Trends Pharm. Sci. 36, 137–144 (2015).
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).
Scott, S. A. et al. Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat. Chem. Biol. 5, 108–117 (2009).
Göbel, K. et al. Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis. Eur. J. Immunol. 44, 2295–2305 (2014).
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).
Lindsley, C. W. & Brown, H. A. Phospholipase D as a therapeutic target in brain disorders. Neuropsychopharmacology 37, 301–302 (2012).
Elvers, M. et al. Impaired αIIbβ3 integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1. Sci. Signal. 3, ra1 (2010).
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).
Monovich, L. et al. Optimization of halopemide for phospholipase D2 inhibition. Bioorg. Med Chem. Lett. 17, 2310–2311 (2007).
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).
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).
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).
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).
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).
Sung, T. C., Zhang, Y., Morris, A. J. & Frohman, M. A. Structural analysis of human phospholipase D1. J. Biol. Chem. 274, 3659–3666 (1999).
Hammond, S. M. et al. Characterization of two alternately spliced forms of phospholipase D1. J. Biol. Chem. 272, 3860–3868 (1997).
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).
Du, G. et al. Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs. J. Cell Biol. 162, 305–315 (2003).
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).
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).
Leiros, I., Secundo, F., Zambonelli, C., Servi, S. & Hough, E. The first crystal structure of a phospholipase D. Structure 8, 655–667 (2000).
Cai, S. & Exton, J. H. Determination of interaction sites of phospholipase D1 for RhoA. Biochem J. 355, 779–785 (2001).
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).
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).
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).
Stuckey, J. A. & Dixon, J. E. Crystal structure of a phospholipase D family member. Nat. Struct. Biol. 6, 278–284 (1999).
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).
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).
Pierce, B. G. et al. ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 30, 1771–1773 (2014).
Henage, L. G., Exton, J. H. & Brown, H. A. Kinetic analysis of a mammalian phospholipase D. J. Biol. Chem. 281, 3408–3417 (2006).
Hicks, S. N. et al. General and versatile autoinhibition of PLC isozymes. Mol. Cell 31, 383–394 (2008).
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).
Lyon, A. M., Begley, J. A., Manett, T. D. & Tesmer, J. J. Molecular mechanisms of phospholipase C β3 autoinhibition. Structure 22, 1844–1854 (2014).
Henage, L. G. Kinetic analysis of a mammalian phospholipase D. J. Biol. Chem. 281, 3408–3417 (2006).
Ali, I. et al. Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae. Acta Crystallogr. F 74, 315–321 (2018).
Cronin, C. N., L, K. B. & Rogers, Joe. Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Sci. 16, 2023–2029 (2007).
Waterman, D. G. et al. Diffraction-geometry refinement in the DIALS framework. Acta Cryst. 72, 558–575 (2016).
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).
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).
Grosse-Kunstleve, R. W. & Adams, P. D. Substructure search procedures for macromolecular structures. Acta Crystallogr. D 59, 1966–1973 (2003).
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).
Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuildwizard. Acta Crystallogr. D 64, 61–69 (2008).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).
Morris, A. J., Frohman, M. A. & Engebrecht, J. Measurement of phospholipase D activity. Anal. Biochem. 252, 1–9 (1997).
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).
Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
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.).
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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.
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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
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DOI: https://doi.org/10.1038/s41589-020-0499-8
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