The signaling molecule Hedgehog (Hh) plays an important role in embryonic patterning and tissue formation by acting as a morphogen that induces discrete cellular states at specific concentration thresholds. These Hh concentration gradients are shaped in part by the interactions of Hh with a new family of proteins that includes Ihog (invertebrates) and CDO (vertebrates). Initial data indicated that Hh binds the first FNIII domain of Ihog (IhogFn1) but that Sonic Hh (Shh) binds the third FNIII domain of CDO (CDOFn3). We initiated biophysical and biochemical studies of these interactions in order to understand the mechanism by which Ihog and CDO influence Hh signaling.

Here we report the crystal structure of the Hh-binding domain of Ihog both alone and in complex with Hh, as well as the structure of Shh bound to CDOFn3. IhogFn1 contains a conserved basic cleft that is capable of binding heparin or heparan sulfate and these molecules are required for Hh binding to IhogFn1 in vitro. Hh binds near this basic cleft on Ihog in the crystal structure, and mutagenesis experiments suggest heparin promotes the binding of Hh to Ihog by binding both molecules simultaneously. Surprisingly, the interaction between Shh and CDOFn3 is not heparin-dependent but instead requires calcium ions. The structure of the complex reveals a previously unknown binuclear calcium-binding site on Shh that is buried at the interface with CDOFn3. Mutagenesis experiments demonstrate that this calcium-binding site is also important for interactions between Shh and Gas1, Hip1 and Ptc. A comparison of the Hh-Ihog structure to that of the Shh-CDO structure reveals that different surfaces on the FNIII domains of Ihog and CDO interact with different surfaces on Hh and Shh, respectively.

These results demonstrate a specific role for heparin or heparan sulfate in the invertebrate Hh signaling pathway and suggest that Hh pathway activity could be modulated by the amount or type of heparan sulfate chains present on the cell surface. In addition, these results explain several naturally occurring missense mutations that disrupt the calcium-binding site in Shh and Indian Hedgehog that lead to the human developmental disorders holoprosencephaly and brachydactyly.