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Comparative Study
. 2008 Oct 16;455(7215):979-83.
doi: 10.1038/nature07358. Epub 2008 Sep 14.

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

Jason S McLellan  1 , Xiaoyan Zheng, Glenn Hauk, Rodolfo Ghirlando, Philip A Beachy, Daniel J Leahy
Affiliations
Comparative Study

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

Jason S McLellan et al. Nature. .

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 responses. 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 cancer. Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle. 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 manner. Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO. 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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Figures

Figure 1
Figure 1. A calcium-binding site on Shh is important for interactions with CDOFn3
a, Sedimentation velocity results for a 1:1 mixture of 20.5 μM ShhN and CDOFn3 in the absence and presence of 1 mM CaCl2. Solid grey lines indicate the experimental sedimentation coefficients for monomeric CDOFn3 and ShhN. The dashed line indicates the sedimentation coefficient calculated in HYDROPRO for the 1:1 complex. b, Semi-transparent molecular surface over a ribbon diagram of ShhN (yellow) bound to CDOFn3 (blue). Calcium and zinc ions are depicted as green and purple spheres, respectively. c, The ShhN-CDOFn3 interface shown in the same orientation as b. The mostly acidic residues on CDOFn3 that interact with mostly basic residues on ShhN are labelled. Semi-transparent surface representations are shown for CDO V918, M919 and I920, which make van der Waal's contacts with Shh H134 and the methylene groups of E90. d, Sidechains in ShhN directly coordinating the two calcium ions are shown in stick representation. Calcium ions are green and three water molecules are shown as red spheres. Hydrogen bonds are depicted as dotted lines. All structure images were rendered with PyMOL (http://www.pymol.org).
Figure 2
Figure 2. Shh binds CDO differently than Hh binds Ihog
a, Schematic of Ihog and CDO. b, Ribbon diagram of the ShhN/CDOFn3 structure superimposed on the HhN/IhogFn1-2 structure by alignment of the Hh molecules. ShhN is colored yellow, CDOFn3 dark blue, HhN red, IhogFn1 light green, and IhogFn2 light blue. Calcium and zinc ions are depicted as green and purple spheres, respectively. The boxed area shows a close-up of the interface between IhogFn1 and the Hh proteins. The sidechains of four ShhN residues changed in the ShhN-Fly mutant are shown as sticks. c, ITC data for: ShhN and CDOFn3 with calcium (left), and HhN and IhogFn1-2 with heparin and calcium (right). d, ITC data for: ShhN and IhogFn1-2 with heparin and calcium (left), and ShhN-Fly and IhogFn1-2 with heparin and EGTA (right).
Figure 3
Figure 3. The Shh calcium-binding site mediates multiple interactions
a, Cells expressing Hip or Ptc were assayed for Shh binding in the presence of 1.8 mM Ca2+ (black bars) and increasing EGTA concentrations (1.8-2.7 mM, white bars). b, Cell-based binding assays were performed with wild-type Shh and a Shh calcium-binding site mutant (Ca Mutant) in the presence of calcium. c, Cell-based binding assays with wild-type Shh were performed in the presence of calcium (black bars) and 0.5-30 μM soluble CDOFn2 (grey bars) or CDOFn3 (white bars). Error bars in a, b and c indicate standard deviation with n = 3. d, Gas1 Fc-fusion protein was immobilized to Protein G beads and binding to ShhN was assayed by a Western blot in the presence and absence of calcium. e, Gas1 pull-down experiments were repeated in the presence of CDOFn3 or CDOFn2.
Figure 4
Figure 4. HPE and BDA1 mutations in Shh and Ihh disrupt binding interactions
a, Loop representation of the mShhN calcium-binding site. Mutation of human Ihh residues E95, D100 and E131 (blue) cause BDA1. Mutation of D88 (purple) in human Shh causes HPE. Calcium ions are depicted as green spheres and hydrogen bonds are shown as dotted lines. b, c, ITC data showing loss of CDOFn3 binding to ShhD88V (b) and IhhE131K (c) in the presence of calcium. d, Normalized binding of Shh and ShhD88V to cells expressing Ptc and Hip. Error bars indicate standard deviation with n = 5. e, Normalized binding of Ihh, IhhE131K and IhhD100E to cells expressing Ptc and Hip. Error bars indicate standard deviation with n = 3.
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References

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