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.

Methods
Cyclopamine (CYC), a steroidal molecule, acts as an agonist or an antagonist for the human Smoothened protein (SMO), depending on where it binds. Here, we investigate the mechanism behind this process, and try to explain the behavior of CYC bound to different sites in SMO. 

Data was collected for each bound pose:

Cyclopamine bound to the orthesteric site present in the Cysteine Rich Domain (CRD) of SMO. (CRD-CYC)
Cyclopamine bound to the allosteric site present in the Transmembrane Domain (TMD) of SMO. (TMD-CYC)
Cyclopamine bound to both the orthosteric and allosteric sites (CRD and TMD) of SMO. (Dual-CYC)

For each system, two starting points were set up as initial systems - corresponding to Active and Inactive SMO. In total, 6 systems were simulated for a total aggregate time of 3 ms of unbiased all-atom simulations. On this Dryad repository, we are uploading the collective variable pickle files calculated from the simulated trajectories, which were then used to analyse the dynamics of CYC-bound SMO. The stripped MD trajectories are available publicly, via Box (https://uofi.app.box.com/s/4g3xmumfmesb68y7tb0fn8wvhvycylrf). The codes used to process the trajectories and the calculated observables are available on Github (https://github.com/ShuklaGroup/SMO_CYC).
Here, we are also providing additional numpy files that were calculated for the MSM construction - the trajectory weights used to reweigh the entire ensemble. 

Molecular Simulations were performed using OpenMM 7.7, and the CHARMM36m force field was used for the simulations.

This work has been performed by Kihong (Max) Kim, Prateek Bansal & Diwakar Shukla at the University of Illinois Urbana-Champaign.