A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Hedgehog (Hh) signaling is initiated by a cholesterol-modified Hh ligand.
• Hh ligand generation involves autoprocessing of the Hh precursor protein.
• Efficient Hh signaling requires coupling of N-S acyl shift and transesterification steps for cholesteroylation.

Purpose of the Study:

• To investigate the role of a conserved aspartate residue (D46) in the Hh autoprocessing domain.
• To elucidate the mechanism by which D46 coordinates the catalytic steps of Hh autoprocessing and cholesteroylation.

Main Methods:

• Site-directed mutagenesis of the D46 residue in the Hh precursor protein.
• Nuclear Magnetic Resonance (NMR) spectroscopy to determine the pKa of D46.
• Assays to measure Hh cholesteroylation and autoprocessing efficiency.

Main Results:

• Mutagenesis revealed D46 suppresses non-native autoprocessing and is essential for cholesterol transesterification.
• NMR data showed D46 has an elevated pKa (5.6) due to hydrogen bonding with a catalytic cysteine.
• A D46N mutation abolished Hh cholesteroylation, indicating the importance of both protonated and deprotonated forms of D46.

Conclusions:

• The aspartate residue D46 acts as a crucial coordinator of Hh autoprocessing.
• D46's proton shuttling mechanism mechanistically links the N-S acyl shift and transesterification steps.
• This coordination by D46 is vital for Hh cholesteroylation and subsequent signal transduction.