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Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP

Nature Structural & Molecular Biology volume 16, pages 698–703 (2009)Cite this article

Abstract

Hedgehog (Hh) morphogens have fundamental roles in development, whereas dysregulation of Hh signaling leads to disease. Multiple cell-surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the Hedgehog-interacting protein (Hhip) is a highly conserved, vertebrate-specific inhibitor of Hh signaling. We have solved a series of crystal structures for the human HHIP ectodomain and Desert hedgehog (DHH) in isolation, as well as HHIP in complex with DHH (HHIP–DHH) and Sonic hedgehog (Shh) (HHIP–Shh), with and without Ca2+. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn2+ and Ca2+ binding sites—functions that may be common to all vertebrate Hh proteins. Zn2+ makes a key contribution to the Hh–HHIP interface, whereas Ca2+ is likely to prevent electrostatic repulsion between the two proteins, suggesting an important modulatory role. This interplay of several metal binding sites suggests a tuneable mechanism for regulation of Hh signaling.

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Figure 1: Structure of the HHIP ectodomain.
Figure 2: Structure of the Shh–HHIP complex.
Figure 3: Binding properties of Hh–HHIP interactions.
Figure 4: Molecular determinants of Hh-receptor interactions.

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References

  1. Ingham, P.W. & McMahon, A.P. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059–3087 (2001).

    Article  CAS  Google Scholar 

  2. Varjosalo, M. & Taipale, J. Hedgehog: functions and mechanisms. Genes Dev. 22, 2454–2472 (2008).

    Article  CAS  Google Scholar 

  3. Roessler, E. et al. Mutations in the human Sonic hedgehog gene cause holoprosencephaly. Nat. Genet. 14, 357–360 (1996).

    Article  CAS  Google Scholar 

  4. Dellovade, T., Romer, J.T., Curran, T. & Rubin, L.L. The hedgehog pathway and neurological disorders. Annu. Rev. Neurosci. 29, 539–563 (2006).

    Article  CAS  Google Scholar 

  5. Yauch, R.L. et al. A paracrine requirement for hedgehog signalling in cancer. Nature 455, 406–410 (2008).

    Article  CAS  Google Scholar 

  6. Mann, R.K. & Beachy, P.A. Novel lipid modifications of secreted protein signals. Annu. Rev. Biochem. 73, 891–923 (2004).

    Article  CAS  Google Scholar 

  7. Zeng, X. et al. A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411, 716–720 (2001).

    Article  CAS  Google Scholar 

  8. Panáková, D., Sprong, H., Marois, E., Thiele, C. & Eaton, S. Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58–65 (2005).

    Article  Google Scholar 

  9. Pepinsky, R.B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998).

    Article  CAS  Google Scholar 

  10. Lum, L. & Beachy, P.A. The Hedgehog response network: sensors, switches, and routers. Science 304, 1755–1759 (2004).

    Article  CAS  Google Scholar 

  11. Wilson, C.W. & Chuang, P.T. New “hogs” in Hedgehog transport and signal reception. Cell 125, 435–438 (2006).

    Article  CAS  Google Scholar 

  12. Beckett, K., Franch-Marro, X. & Vincent, J.P. Glypican-mediated endocytosis of Hedgehog has opposite effects in flies and mice. Trends Cell Biol. 18, 360–363 (2008).

    Article  CAS  Google Scholar 

  13. Chuang, P.T. & McMahon, A.P. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397, 617–621 (1999).

    Article  CAS  Google Scholar 

  14. Kang, J.S., Zhang, W. & Krauss, R.S. Hedgehog signaling: cooking with Gas1. Sci. STKE 2007, pe50 (2007).

    Article  Google Scholar 

  15. Coulombe, J., Traiffort, E., Loulier, K., Faure, H. & Ruat, M. Hedgehog interacting protein in the mature brain: membrane-associated and soluble forms. Mol. Cell. Neurosci. 25, 323–333 (2004).

    Article  CAS  Google Scholar 

  16. Parmantier, E. et al. Schwann cell-derived Desert hedgehog controls the development of peripheral nerve sheaths. Neuron 23, 713–724 (1999).

    Article  CAS  Google Scholar 

  17. Bourikas, D. et al. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8, 297–304 (2005).

    Article  CAS  Google Scholar 

  18. Olsen, C.L., Hsu, P.P., Glienke, J., Rubanyi, G.M. & Brooks, A.R. Hedgehog-interacting protein is highly expressed in endothelial cells but down-regulated during angiogenesis and in several human tumors. BMC Cancer 4, 43 (2004).

    Article  Google Scholar 

  19. Tada, M. et al. Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma. Clin. Cancer Res. 14, 3768–3776 (2008).

    Article  CAS  Google Scholar 

  20. Taniguchi, H. et al. Intrahepatic mRNA levels of type I interferon receptor and interferon-stimulated genes in genotype 1b chronic hepatitis C. Association between IFNAR1 mRNA level and sustained response to interferon therapy. Intervirology 50, 32–39 (2007).

    Article  CAS  Google Scholar 

  21. Tojo, M., Kiyosawa, H., Iwatsuki, K. & Kaneko, F. Expression of a sonic hedgehog signal transducer, hedgehog-interacting protein, by human basal cell carcinoma. Br. J. Dermatol. 146, 69–73 (2002).

    Article  CAS  Google Scholar 

  22. Aricescu, A.R., Lu, W. & Jones, E.Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243–1250 (2006).

    Article  Google Scholar 

  23. Harding, M.M. Geometry of metal-ligand interactions in proteins. Acta Crystallogr. D Biol. Crystallogr. 57, 401–411 (2001).

    Article  CAS  Google Scholar 

  24. Hall, T.M., Porter, J.A., Beachy, P.A. & Leahy, D.J. A potential catalytic site revealed by the 1.7-crystal structure of the amino-terminal signalling domain of Sonic hedgehog. Nature 378, 212–216 (1995).

    Article  CAS  Google Scholar 

  25. Day, E.S. et al. Zinc-dependent structural stability of human Sonic hedgehog. Biochemistry 38, 14868–14880 (1999).

    Article  CAS  Google Scholar 

  26. McLellan, J.S. et al. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455, 979–983 (2008).

    Article  CAS  Google Scholar 

  27. Fuse, N. et al. Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for patched. Proc. Natl. Acad. Sci. USA 96, 10992–10999 (1999).

    Article  CAS  Google Scholar 

  28. Orioli, I.M. et al. Identification of novel mutations in SHH and ZIC2 in a South American (ECLAMC) population with holoprosencephaly. Hum. Genet. 109, 1–6 (2001).

    Article  CAS  Google Scholar 

  29. Ribeiro, L.A. & Richieri-Costa, A. Single median maxillary central incisor, hypophyseal tumor, and SHH mutation. Am. J. Med. Genet. A. 136A, 346–347 (2005).

    Article  Google Scholar 

  30. Hofer, A.M. & Brown, E.M. Extracellular calcium sensing and signalling. Nat. Rev. Mol. Cell Biol. 4, 530–538 (2003).

    Article  CAS  Google Scholar 

  31. Springer, T.A., Zhu, J. & Xiao, T. Structural basis for distinctive recognition of fibrinogen γC peptide by the platelet integrin αIIbβ3. J. Cell Biol. 182, 791–800 (2008).

    Article  CAS  Google Scholar 

  32. Chang, V.T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

    Article  CAS  Google Scholar 

  33. Walter, T.S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D Biol. Crystallogr. 61, 651–657 (2005).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  36. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).

    CAS  Google Scholar 

  37. Terwilliger, T.C. Automated side-chain model building and sequence assignment by template matching. Acta Crystallogr. D Biol. Crystallogr. 59, 45–49 (2003).

    Article  Google Scholar 

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

    Article  Google Scholar 

  39. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  Google Scholar 

  40. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  42. 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).

    Article  Google Scholar 

  43. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  44. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

  45. Hooft, R.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    Article  CAS  Google Scholar 

  46. Stuart, D.I., Levine, M., Muirhead, H. & Stammers, D.K. Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6. J. Mol. Biol. 134, 109–142 (1979).

    Article  CAS  Google Scholar 

  47. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Trombetta, E.S. & Parodi, A.J. Quality control and protein folding in the secretory pathway. Annu. Rev. Cell Dev. Biol. 19, 649–676 (2003).

    Article  CAS  Google Scholar 

  50. O'Callaghan, C.A. et al. BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal. Biochem. 266, 9–15 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the staff of European Synchrotron Radiation Facility beamline ID 29 and Diamond beamlines I02 and I03 for assistance with data collection, T. Walter for help with crystallization, G. Sutton for help with MALS experiments, and S. Graham and D. Stuart for discussions. We acknowledge the use of the crystallization facilities provided by the Medical Research Council (MRC)–funded Oxford Protein Production Facility (OPPF). The work was funded by the Wellcome Trust. A.R.A. and C.A.O'C. are funded by the MRC. E.Y.J. is funded by Cancer Research UK. C.S. is funded by the Wellcome Trust.

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Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Benjamin Bishop, A Radu Aricescu, Karl Harlos, E Yvonne Jones & Christian Siebold

  2. Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK

    Chris A O'Callaghan

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  1. Benjamin Bishop
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Contributions

C.S. designed the project; B.B. and C.S. produced the constructs and crystallized the proteins; B.B. and C.A.O'C. performed the SPR experiments; K.H. and C.S. collected X-ray data; C.S. solved the structures; B.B, A.R.A., C.A.O'C., E.Y.J. and C.S. analyzed the data and wrote the paper.

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Correspondence to Christian Siebold.

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Bishop, B., Aricescu, A., Harlos, K. et al. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat Struct Mol Biol 16, 698–703 (2009). https://doi.org/10.1038/nsmb.1607

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