Dual roles of the sterol recognition region in Hedgehog protein modification

Abstract

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.

Hedgehog proteins create a dynamic cholesterol interface

“…This study provided a detailed picture of the Hint fold active site during the cholesterolysis process, and suggested SRR residues that might influence the efficiency of the reaction. However, this model was generated in the absence of a membrane, and SRR residues that featured prominently in the fly model had little significance in our hSHH cholesterolysis experiments [ 17 ]. We therefore set out to construct an atomistic model of the human Sonic hedgehog Hog domain that incorporated our experimental results.…”

“…Notably, studies on the hydrolysis of self-splicing inteins show that the Hint fold is largely unchanged during the splicing process, supporting the use of this template for our pre-cleavage model [ 36 – 39 ]. To evaluate ab initio structure predictions for the SRR, which has no homologues in the PDB, we relied on our biochemical and cellular analysis of hSHH SRR reactivity [ 17 ]. We eliminated structures that lacked α-helices that we had previously characterized by circular dichroism analysis (1 st SRR helix: W372-L390; 2 nd SRR helix: I432-L447).…”

“…Notably, the 1 st SRR helix remained firmly anchored to the surface of the phospholipid bilayer throughout the simulations. Our previous studies showed that the 1 st SRR helix in hSHH contains a conserved HPR motif (H374, P379, R381) succeeded by three leucine residues (L382, L386, L390) that are critical for hSHH cholesterolysis in cells ( Fig 2C ) [ 17 ]. The requirement for these leucine residues in cellular but not biochemical activity suggests that they might play a role in membrane association.…”

“…hSHH cloning, protein expression, and Western blot analysis were performed according to previously described methods [ 17 ]. Wild type hSHH (residues 1–462) and mutants were cloned in the pCMV6 vector (Origene RC222175) with a stop codon inserted before the C-terminal Myc-DDK tag (see S1 Table ).…”

“…Our recent alanine scanning studies have identified residues in the SRR that reduce hSHH cholesterolysis and control the localization of SRR fusion proteins in cells [ 17 ], suggesting that the SRR plays roles in both processes. However, the structure of the membrane-associated Hog (Hint-SRR) structure that catalyzes cholesterolysis remains enigmatic.…”