Binding Position Dependent Modulation of Smoothened Activity by Cyclopamine.

Cyclopamine is a natural alkaloid that is known to act as an agonist when it binds to the Cysteine Rich Domain (CRD) of the Smoothened receptor and as an antagonist when it binds to the Transmembrane Domain (TMD). To study the effect of cyclopamine binding to each binding site experimentally, mutations in the other site are required. Hence, simulations are critical for understanding the WT activity due to binding at different sites. Additionally, there is a possibility that cyclopamine could bind to both sites simultaneously especially at high concentration, the implications of which remain unknown. We performed three independent sets of simulations to observe the receptor activation with cyclopamine bound to each site independently (CRD, TMD) and bound to both sites simultaneously. Using multi-milliseconds long aggregate MD simulations combined with Markov state models and machine learning, we explored the dynamic behavior of cyclopamine's interactions with different domains of WT SMO. A higher population of the active state at equilibrium, a lower activation free energy barrier of ~ 2 kcal/mol, and expansion of the hydrophobic tunnel to facilitate cholesterol transport agrees with the cyclopamine's agonistic behavior when bound to the CRD of SMO. A higher population of the inactive state at equilibrium, a higher free energy barrier of ~ 4 kcal/mol and restricted the hydrophobic tunnel to impede cholesterol transport showed cyclopamine's antagonistic behavior when bound to TMD. With cyclopamine bound to both sites, there was a slightly larger inactive population at equilibrium and an increased free energy barrier (~ 3.5 kcal/mol). The tunnel was slightly larger than when solely bound to TMD, and showed a balance between agonism and antagonism with respect to residue movements exhibiting an overall weak antagonistic effect.