Binding Position Dependent Modulation of Smoothened Activity by Cyclopamine.

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• Cyclopamine's dual role as agonist and antagonist at Smoothened receptor (SMO) binding sites (Cysteine Rich Domain - CRD, Transmembrane Domain - TMD) necessitates understanding its precise molecular interactions.
• Investigating cyclopamine's simultaneous binding to both CRD and TMD is crucial for elucidating its full functional spectrum.

Approach:

• Utilized multi-millisecond aggregate Molecular Dynamics (MD) simulations to analyze wild-type SMO (WT SMO) interactions with cyclopamine.
• Integrated Markov state models and machine learning to explore dynamic behaviors and free energy landscapes.
• Performed simulations with cyclopamine bound to CRD, TMD, and both sites concurrently.

Key Points:

• CRD binding: Promotes active SMO state, lowers activation free energy (~2 kcal/mol), and expands the hydrophobic tunnel for cholesterol transport, consistent with agonism.
• TMD binding: Favors inactive SMO state, increases activation free energy (~4 kcal/mol), and restricts the hydrophobic tunnel, indicating antagonism.
• Dual binding: Results in a slightly higher inactive population, increased free energy barrier (~3.5 kcal/mol), and a net weak antagonistic effect with balanced residue movements.

Conclusions:

• Computational simulations provide critical insights into cyclopamine's context-dependent activity at SMO.
• Distinct binding modes at CRD and TMD dictate SMO activation and cholesterol transport.
• Simultaneous CRD-TMD binding modulates SMO activity with a predominantly weak antagonistic outcome.