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Crystal structure of autotaxin and insight into GPCR activation by lipid mediators

Nature Structural & Molecular Biology volume 18, pages 205–212 (2011)Cite this article

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Abstract

Autotaxin (ATX, also known as Enpp2) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine to generate lysophosphatidic acid (LPA), a lipid mediator that activates G protein–coupled receptors to evoke various cellular responses. Here, we report the crystal structures of mouse ATX alone and in complex with LPAs with different acyl-chain lengths and saturations. These structures reveal that the multidomain architecture helps to maintain the structural rigidity of the lipid-binding pocket, which accommodates the respective LPA molecules in distinct conformations. They indicate that a loop region in the catalytic domain is a major determinant for the substrate specificity of the Enpp family enzymes. Furthermore, along with biochemical and biological data, these structures suggest that the produced LPAs are delivered from the active site to cognate G protein–coupled receptors through a hydrophobic channel.

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Figure 1: Overall architecture.
Figure 2: Active site.
Figure 3: Interdomain interaction.
Figure 4: Structural determinant of the substrate specificity.
Figure 5: Biochemical characterization of the wild-type and mutant ATXs.
Figure 6: Hydrophobic channel.

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References

  1. Stracke, M.L. et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J. Biol. Chem. 267, 2524–2529 (1992).

    CAS  PubMed  Google Scholar 

  2. Umezu-Goto, M. et al. Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production. J. Cell Biol. 158, 227–233 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Tokumura, A. et al. Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J. Biol. Chem. 277, 39436–39442 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Noguchi, K., Herr, D., Mutoh, T. & Chun, J. Lysophosphatidic acid (LPA) and its receptors. Curr. Opin. Pharmacol. 9, 15–23 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Moolenaar, W.H., van Meeteren, L.A. & Giepmans, B.N. The ins and outs of lysophosphatidic acid signaling. Bioessays 26, 870–881 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Contos, J.J., Fukushima, N., Weiner, J.A., Kaushal, D. & Chun, J. Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior. Proc. Natl. Acad. Sci. USA 97, 13384–13389 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tanaka, M. et al. Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid. J. Biol. Chem. 281, 25822–25830 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. van Meeteren, L.A. et al. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Mol. Cell. Biol. 26, 5015–5022 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kanda, H. et al. Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs. Nat. Immunol. 9, 415–423 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Inoue, M. et al. Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling. Nat. Med. 10, 712–718 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Liu, S. et al. Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell 15, 539–550 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yang, S.Y. et al. Expression of autotaxin (NPP-2) is closely linked to invasiveness of breast cancer cells. Clin. Exp. Metastasis 19, 603–608 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Stassar, M.J. et al. Identification of human renal cell carcinoma associated genes by suppression subtractive hybridization. Br. J. Cancer 85, 1372–1382 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Baumforth, K.R. et al. Induction of autotaxin by the Epstein-Barr virus promotes the growth and survival of Hodgkin lymphoma cells. Blood 106, 2138–2146 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Zhang, G., Zhao, Z., Xu, S., Ni, L. & Wang, X. Expression of autotaxin mRNA in human hepatocellular carcinoma. Chin. Med. J. 112, 330–332 (1999).

    CAS  PubMed  Google Scholar 

  16. Masuda, A. et al. Serum autotaxin measurement in haematological malignancies: a promising marker for follicular lymphoma. Br. J. Haematol. 143, 60–70 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Kishi, Y. et al. Autotaxin is overexpressed in glioblastoma multiforme and contributes to cell motility of glioblastoma by converting lysophosphatidylcholine to lysophosphatidic acid. J. Biol. Chem. 281, 17492–17500 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Hama, K. et al. Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1 . J. Biol. Chem. 279, 17634–17639 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Parrill, A.L. & Baker, D.L. Autotaxin inhibition: challenges and progress toward novel anti-cancer agents. Anticancer Agents Med. Chem. 8, 917–923 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Stefan, C., Jansen, S. & Bollen, M. NPP-type ectophosphodiesterases: unity in diversity. Trends Biochem. Sci. 30, 542–550 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Cimpean, A., Stefan, C., Gijsbers, R., Stalmans, W. & Bollen, M. Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2. Biochem. J. 381, 71–77 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tabata, S. et al. A rapid screening method for cell lines producing singly tagged recombinant proteins using the 'TARGET tag' system. J. Proteomics 73, 1777–1785 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Jansen, S. et al. Proteolytic maturation and activation of autotaxin (NPP2), a secreted metastasis-enhancing lysophospholipase D. J. Cell Sci. 118, 3081–3089 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Zhou, A., Huntington, J.A., Pannu, N.S., Carrell, R.W. & Read, R.J. How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat. Struct. Biol. 10, 541–544 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Deng, G., Royle, G., Wang, S., Crain, K. & Loskutoff, D.J. Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin. J. Biol. Chem. 271, 12716–12723 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. van Meeteren, L.A. & Moolenaar, W.H. Regulation and biological activities of the autotaxin–LPA axis. Prog. Lipid Res. 46, 145–160 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Lee, H.Y. et al. Stimulation of tumor cell motility linked to phosphodiesterase catalytic site of autotaxin. J. Biol. Chem. 271, 24408–24412 (1996).

    Article  CAS  PubMed  Google Scholar 

  28. Clair, T. et al. Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate. Cancer Res. 63, 5446–5453 (2003).

    CAS  PubMed  Google Scholar 

  29. Gijsbers, R., Aoki, J., Arai, H. & Bollen, M. The hydrolysis of lysophospholipids and nucleotides by autotaxin (NPP2) involves a single catalytic site. FEBS Lett. 538, 60–64 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Gijsbers, R., Ceulemans, H., Stalmans, W. & Bollen, M. Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases. J. Biol. Chem. 276, 1361–1368 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Zalatan, J.G., Fenn, T.D., Brunger, A.T. & Herschlag, D. Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: implications for mechanism and evolution. Biochemistry 45, 9788–9803 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Dennis, J. et al. Phosphodiesterase-Iα/autotaxin's MORFO domain regulates oligodendroglial process network formation and focal adhesion organization. Mol. Cell. Neurosci. 37, 412–424 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Ghosh, M., Meiss, G., Pingoud, A., London, R.E. & Pedersen, L.C. Structural insights into the mechanism of nuclease A, a ββα metal nuclease from Anabaena. J. Biol. Chem. 280, 27990–27997 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Jansen, S. et al. An essential oligomannosidic glycan chain in the catalytic domain of autotaxin, a secreted lysophospholipase-D. J. Biol. Chem. 282, 11084–11091 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Jansen, S., Andries, M., Derua, R., Waelkens, E. & Bollen, M. Domain interplay mediated by an essential disulfide linkage is critical for the activity and secretion of the metastasis-promoting enzyme autotaxin. J. Biol. Chem. 284, 14296–14302 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sakagami, H. et al. Biochemical and molecular characterization of a novel choline-specific glycerophosphodiester phosphodiesterase belonging to the nucleotide pyrophosphatase/phosphodiesterase family. J. Biol. Chem. 280, 23084–23093 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. van Meeteren, L.A. et al. Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate. J. Biol. Chem. 280, 21155–21161 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Winkler, F.K., D'Arcy, A. & Hunziker, W. Structure of human pancreatic lipase. Nature 343, 771–774 (1990).

    Article  CAS  PubMed  Google Scholar 

  39. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  41. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  43. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  PubMed  Google Scholar 

  44. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tsuda, S. et al. Cyclic phosphatidic acid is produced by autotaxin in blood. J. Biol. Chem. 281, 26081–26088 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to the beam-line staffs at NW12A and NE3A of KEK PF-AR and BL41XU of SPring-8 for assistance in data collection. We thank L. Bonnefond (University of Tokyo, Japan) for critical reading of the manuscript. This work was supported by a grant for the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to O.N. and J.A., by a National Institute of Biomedical Innovation (Japan) grant to J.A. and by MEXT grants to R.I. and O.N.

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Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Hiroshi Nishimasu, Ryuichiro Ishitani & Osamu Nureki

  2. Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan

    Shinichi Okudaira, Kotaro Hama, Asuka Inoue & Junken Aoki

  3. Institute for Protein Research, Osaka University, Osaka, Japan

    Emiko Mihara & Junichi Takagi

  4. Biomolecular Characterization Team and CREST/JST, RIKEN, Saitama, Japan

    Naoshi Dohmae

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  1. Hiroshi Nishimasu
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Contributions

H.N. crystallized and determined the structure of ATX and wrote the paper; S.O., K.H. and A.I. conducted biochemical and cell biological analyses of the wild-type and mutant ATXs; E.M. and J.T. prepared the recombinant ATX proteins; A.I., N.D. and J.A. did the mass spectrometry analysis of endogenous LPA; R.I. assisted with the structural determination; and O.N. designed the work and also assisted with the structure determination. All authors discussed the results and commented on the manuscript; O.N., J.A. and J.T. supervised the work.

Corresponding authors

Correspondence to Junichi Takagi, Junken Aoki or Osamu Nureki.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Discussion (PDF 4596 kb)

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Nishimasu, H., Okudaira, S., Hama, K. et al. Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. Nat Struct Mol Biol 18, 205–212 (2011). https://doi.org/10.1038/nsmb.1998

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