In Vitro Reconstitution of Vertebrate Sonic Hedgehog Protein Cholesterolysis; Native and Engineered Sterol Specificity

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on release from a precursor protein through peptide-bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we first describe the in vitro reconstitution of SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multiwell plates by monitoring changes in fluorescence resonance energy transfer (FRET) from a bacterially expressed, engineered precursor construct. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol (KM, 1–2 μM, cholesterolysis t1/2 of ∼11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 μM, t1/2 > 10 h). Next, screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we observed a shared preference for the zwitterionic n-dodecylphosphocholine (DPC, Fos-choline-12), while finding that cationic detergents appeared incompatible with cholesterolysis. Lastly, we report that an alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC enables engineering of substrate specificity away from cholesterol and toward rationally designed hypernucleophilic sterols via chemical rescue. Of those sterols, 2-β-carboxycholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established a FRET-based kinetic assay to continuously monitor enzymatic activity of vertebrate SHhC domains, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis, while also identifying the first SHhC mutant/sterol pairing for orthogonal sterolysis.