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The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla

Nature volume 455, pages 979–983 (2008)Cite this article

Abstract

Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses1,2. Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer3,4,5,6,7. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle8,9,10. Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparin-dependent manner12,13. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO12,14. Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN–CDO interaction is completely unlike the HhN–Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.

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Figure 1: A calcium-binding site on Shh is important for interactions with CDOFn3.
Figure 2: Shh binds CDO differently than Hh binds Ihog.
Figure 3: The Shh calcium-binding site mediates multiple interactions.
Figure 4: HPE and BDA1 mutations in Shh and Ihh disrupt binding interactions.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates for the Shh–CDOFn3 complex have been deposited in the Protein Data Bank with accession number 3D1M.

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Acknowledgements

We thank R. Abramowitz and J. Schwanof of beamline X4C at the National Synchroton Light Source for assistance with X-ray data collection, C. W. Vander Kooi for suggesting ions may promote Shh–CDO interactions, W. Yang, J. Nathans, W. I. Weis, K. C. Garcia, P. A. Cole and L. M. Amzel for comments on the manuscript, and D. Chan and K. Cheah for communicating results before publication. We thank A. P. McMahon and C. M. Fan for the mouse Hip1 and mouse Gas1–Fc complementary DNAs, respectively. This research was supported in part by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (R.G.). D.J.L. is supported by R01 HD055545 and P.A.B. is an HHMI investigator. J.S.M. is supported by a National Science Foundation Graduate Research Fellowship. X.Z. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-1915-06).

Author information

Author notes

  1. Jason S. McLellan and Xiaoyan Zheng: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA,

    Jason S. McLellan, Glenn Hauk & Daniel J. Leahy

  2. Department of Developmental Biology, and,

    Xiaoyan Zheng & Philip A. Beachy

  3. Howard Hughes Medical Institute Stanford University School of Medicine, Stanford, California 94305, USA ,

    Philip A. Beachy

  4. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland 20892, USA ,

    Rodolfo Ghirlando

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  1. Jason S. McLellan
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Correspondence to Daniel J. Leahy.

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McLellan, J., Zheng, X., Hauk, G. et al. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455, 979–983 (2008). https://doi.org/10.1038/nature07358

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