Nature provides a number of mechanisms to encode dynamic information in biomolecules. In metazoans, there exist rare chemical modifications that occur in entirely unique regimes. One such example occurs in the Hedgehog (Hh) morphogens, proteins singular across all domains of life for the nature of their covalent ligation to cholesterol. The isoform-and context-specific efficiency of this ligation profoundly impacts the activity of Hh morphogens and represents an unexplored facet of Hh ligand-dependent cancers. To elucidate the chemical mechanism of this modification, we have defined roles of the uncharacterized sterol recognition region (SRR) in Hh proteins. We use a combination of sequence conservation, directed mutagenesis, and biochemical assays to specify residues of the SRR participate in cellular and biochemical aspects of Hh cholesterolysis. Our investigations offer a functional portrait of this region, providing opportunities to identify parallel reactivity in nature and a template to design tools in chemical biology.

Nature provides a number of mechanisms to encode dynamic information in biomolecules. In metazoans, there exist rare chemical modifications that occur through entirely unique mechanistic regimes. One such example occurs in the Hedgehog (Hh) morphogens, proteins singular across all domains of life for the nature of their covalent ligation to cholesterol. The isoform-and context-specific efficiency of the ligation reaction has profound impact on the activity of Hh morphogens and represents an unexplored aspect of Hh ligand-dependent cancers. To elucidate the chemical mechanism of this modification, we have defined roles of the uncharacterized sterol recognition region (SRR) in Hh proteins. We use a combination of sequence conservation, directed mutagenesis, and biochemical assays to specify residues of the SRR that are responsible for cellular and biochemical processes in Hh cholesterolysis. Our investigations offer the first functional template of this region, providing opportunities to identify parallel reactivity in nature and revealing new mechanisms that can be exploited as tools in chemical biology.

In the original published version of the article, an error was introduced into Fig. 1a during the typesetting process. The error has been corrected in the PDF and HTML versions of the paper.

The Hedgehog (Hh) signaling pathway plays an essential role during embryogenesis and when dysregulated, can lead to human cancer (basal cell carcinomas) and the most lethal form of juvenile medulloblastoma. Binding of the Hh ligand to its receptor Patched (Ptch) relieves inhibition of the GPCR protein Smoothened (Smo) leading to activation of the Gli transcription factors and changes in target gene expression. The Hh ligand is decorated with two covalent modifications, N‐terminal palmitoylation and C‐terminal cholesterolyation. Hh proteins are synthesized as approximately 45 kDa full‐length species, whereupon a unique process yields the mature morphogen: an autocatalytic cleavage that splits the protein in two and attaches cholesterol to the newly formed C‐terminus. Of the two approximately equal molecular weight cleavage products, the dually lipidated N‐terminal fragment (Hh‐N/“Hog” domain) constitutes the classical Hh morphogen, whereas the C‐terminus (Hh‐C/“Hint”) is responsible for the cleavage reaction. In order to elucidate the chemical mechanism of cholesterolyation, we have determined the roles of the previously uncharacterized sterol recognition region (SRR) in Hh proteins. Using bioinformatics, alanine scanning, and biochemical assays we have identified key motifs and specified residues of the SRR that are required for biochemical processing of Hh. These results provide a first in detail insights into the role of this unique small region of the Hh protein that is capable of cellular targeting and engaging cholesterol to generate an active Hh ligand. Support or Funding Information This work was supported by the Margaret E. Early Medical Research Trust.