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

Hedgehog (Hh) signaling molecules are involved in multiple developmental processes. Hedgehog-interacting protein (Hhip) binds and inhibits vertebrate Hh proteins. Structures of HHIP in complex with SHH and DHH now show a distinct binding site from previous ligand structures, with metal-binding sites having a role in interaction. 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 Ca 2+ . The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn 2+ and Ca 2+ binding sites—functions that may be common to all vertebrate Hh proteins. Zn 2+ makes a key contribution to the Hh–HHIP interface, whereas Ca 2+ 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.