Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric G_i

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• The Hedgehog signaling pathway is crucial for development and cancer, mediated by the Smoothened (SMO) G-protein-coupled receptor (GPCR).
• Mechanisms of SMO activation, its interaction with Patched-1 (PTCH1), and G-protein coupling remained largely unknown.
• Understanding these processes is vital for targeting the pathway in diseases.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying Hedgehog signal transduction.
• To identify endogenous ligands that modulate SMO activity.
• To determine how SMO interacts with heterotrimeric G proteins and activates downstream signaling.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structure of human SMO.
• Biochemical assays were performed to assess G-protein signaling in vitro.
• Identification of endogenous ligands for PTCH1.

Main Results:

• 24,25-epoxycholesterol was identified as an endogenous ligand of PTCH1, capable of stimulating Hedgehog signaling.
• A cryo-EM structure revealed 24(S),25-epoxycholesterol bound to human SMO in complex with a heterotrimeric G_i protein.
• The structure elucidated a novel G_i-coupled activation mechanism for SMO, distinct from class-A GPCRs.

Conclusions:

• 24,25-epoxycholesterol is a key molecule in Hedgehog pathway activation.
• The study provides the first structural insights into the G-protein coupling and activation of a class-F GPCR (SMO).
• This work advances our understanding of fundamental signal transduction and offers potential therapeutic targets.