Structural basis for EGFR ligand sequestration by Argos

Abstract

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes1,2. Inappropriate activation of these receptors is a key feature of many human cancers3, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila4. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR5. Here we describe the 1.6-Å resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations4,6, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold7) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-β family receptors8. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA9. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.

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Figure 1: Structure of the Argos–Spitz complex.
Figure 2: Argos has three similar domains that resemble the three-finger toxin fold of TGF-β receptors.
Figure 3: Spitz-binding interactions.
Figure 4: Argos, EGFR and structural homologues entrap the EGF domain with two binding sites.

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References

  1. 1

    Holbro, T. & Hynes, N. E. ErbB receptors: directing key signaling networks throughout life. Annu. Rev. Pharmacol. Toxicol. 44, 195–221 (2004)

  2. 2

    Shilo, B. Z. Regulating the dynamics of EGF receptor signaling in space and time. Development 132, 4017–4027 (2005)

  3. 3

    Hynes, N. E. & Lane, H. A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nature Rev. Cancer 5, 341–354 (2005)

  4. 4

    Freeman, M., Klambt, C., Goodman, C. S. & Rubin, G. M. The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69, 963–975 (1992)

  5. 5

    Klein, D. E., Nappi, V. M., Reeves, G. T., Shvartsman, S. Y. & Lemmon, M. A. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration. Nature 430, 1040–1044 (2004)

  6. 6

    Kretzschmar, D. et al. giant lens, a gene involved in cell determination and axon guidance in the visual system of Drosophila melanogaster . EMBO J. 11, 2531–2539 (1992)

  7. 7

    Tsetlin, V. Snake venom α-neurotoxins and other ‘three-finger’ proteins. Eur. J. Biochem. 264, 281–286 (1999)

  8. 8

    Greenwald, J., Fischer, W. H., Vale, W. W. & Choe, S. Three-finger toxin fold for the extracellular ligand-binding domain of the type II activin receptor serine kinase. Nature Struct. Biol. 6, 18–22 (1999)

  9. 9

    Barinka, C. et al. Structural basis of interaction between urokinase-type plasminogen activator and its receptor. J. Mol. Biol. 363, 482–495 (2006)

  10. 10

    Dauter, Z., Dauter, M. & Rajashankar, K. R. Novel approach to phasing proteins: derivatization by short cryo-soaking with halides. Acta Crystallogr. D Biol. Crystallogr. 56, 232–237 (2000)

  11. 11

    Zhang, C. & Kim, S.-H. The anatomy of protein β-sheet topology. J. Mol. Biol. 299, 1075–1089 (2000)

  12. 12

    Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993)

  13. 13

    Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004)

  14. 14

    Allendorph, G. P., Vale, W. W. & Choe, S. Structure of the ternary signaling complex of a TGF-β superfamily member. Proc. Natl Acad. Sci. USA 103, 7643–7648 (2006)

  15. 15

    Alvarado, D., Evans, T. A., Sharma, R., Lemmon, M. A. & Duffy, J. B. Argos mutants define an affinity threshold for spitz inhibition in vivo . J. Biol. Chem. 281, 28993–29001 (2006)

  16. 16

    Garrett, T. P. J. et al. Crystal structure of a truncated epidermal growth factor receptor extracellular domain bound to transforming growth factor α. Cell 110, 763–773 (2002)

  17. 17

    Ogiso, H. et al. Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110, 775–787 (2002)

  18. 18

    Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

  19. 19

    Huai, Q. et al. Structure of human urokinase plasminogen activator in complex with its receptor. Science 311, 656–659 (2006)

  20. 20

    Rösel, M., Claas, C., Seiter, S., Herlevsen, M. & Zöller, M. Cloning and functional characterization of a new phosphatidyl-inositol anchored molecule of a metastasizing rat pancreatic tumor. Oncogene 17, 1989–2002 (1998)

  21. 21

    Temerinac, S. et al. Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera. Blood 95, 2569–2576 (2000)

  22. 22

    Hansen, L. V., Laerum, O. D., Illemann, M., Nielsen, B. S. & Ploug, M. Altered expression of the urokinase receptor homologue, C4.4A, in invasive areas of human esophageal squamous cell carcinoma. Int. J. Cancer 122, 734–741 (2008)

  23. 23

    Kenny, P. A. & Bissell, M. J. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J. Clin. Invest. 117, 337–345 (2007)

  24. 24

    Zhou, B. B. et al. Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non-small cell lung cancer. Cancer Cell 10, 39–50 (2006)

  25. 25

    Fujimoto, N. et al. High expression of ErbB family members and their ligands in lung adenocarcinomas that are sensitive to inhibition of epidermal growth factor receptor. Cancer Res. 65, 11478–11485 (2005)

  26. 26

    Hynes, N. E. & Schlange, T. Targeting ADAMS and ERBBs in lung cancer. Cancer Cell 10, 7–11 (2006)

  27. 27

    Borrell-Pagès, M., Rojo, F., Albanell, J., Baselga, J. & Arribas, J. TACE is required for the activation of the EGFR by TGF-α in tumors. EMBO J. 22, 1114–1124 (2003)

  28. 28

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

  29. 29

    CCP4 (Collaborative Computational Project Number 4). The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994)

  30. 30

    Lu, H. S. et al. Crystal structure of human epidermal growth factor and its dimerization. J. Biol. Chem. 276, 34913–34917 (2001)

  31. 31

    Miura, G. I. et al. Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting its diffusion. Dev. Cell 10, 167–176 (2006)

  32. 32

    Iwaki, T., Figuera, M., Ploplis, V. A. & Castellino, F. J. Rapid selection of Drosophila S2 cells with the puromycin resistance gene. Biotechniques 35, 482–486 (2003)

  33. 33

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  34. 34

    Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Cryst. 37, 843–844 (2004)

  35. 35

    Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002)

  36. 36

    McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005)

  37. 37

    Hayward, S. & Lee, R. A. Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. J. Mol. Graph. Model. 21, 181–183 (2002)

  38. 38

    DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, CA, 2002)

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Acknowledgements

We thank members of the Lemmon and Ferguson laboratories, G. Van Duyne and J. Shorter for advice and critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (to M.A.L.) and the US Army Breast Cancer Research Program (to D.E.K. and M.A.L.).

Author Contributions D.E.K. and M.A.L. conceived and designed the project. D.E.K. was responsible for all construct design and execution of protein biochemistry, crystallization, and data collection. D.E.K. solved and refined the Argos217–SpitzEGF complex structure. S.E.S. solved and refined the structures of uncomplexed Argos217 and SpitzEGF by molecular replacement using datasets collected by D.E.K. K.N. helped with crystal manipulation and data collection. F.S. performed binding studies with Argos and Spitz variants, as well as analytical ultracentrifugation, directed by D.E.K. D.E.K. and M.A.L. interpreted data and wrote the manuscript.

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Correspondence to Mark A. Lemmon.

Additional information

Coordinates have been deposited in the Protein Data Bank under codes 3CA7 (SpitzEGF), 3C9A (Argos217–SpitzEGF complex), and 3CGU (Argos217 alone).

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Supplementary Information

The file contains Supplementary Discussion, Supplementary Figures S1-S7, and Supplementary Table 1 (PDF 5693 kb)

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Klein, D., Stayrook, S., Shi, F. et al. Structural basis for EGFR ligand sequestration by Argos. Nature 453, 1271–1275 (2008) doi:10.1038/nature06978

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