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...., 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 o..., , # Binding Position Dependent Modulation of Smoothened Activity by Cyclopamine

[https://doi.org/10.5061/dryad.4b8gthtmf](https://doi.org/10.5061/dryad.4b8gthtmf)

The following is an explanation of the overall dataset submitted to this repository:

1. Stripped_Parm.tar.gz (2.69 MB): parameter files used for the simulations. Files are in AMBER22 format.
2. Dtraj_Weights.tar.gz (15.57 MB): Trajectory weights obtained after MSM construction. These were then used to reweigh the ensembles shown in Figures 3, 4, 5, 6.
* To construct the MSM, a set of 57 distances was chosen (described in the manuscript). time-lagged Independent Component Analysis (tICA) was performed on these set of distances, which uses a linear combination of the 57 distances to reduce the dimensions, where the first dimension represented the slowest kinetic process (activation) observed in the system. Then, k-means clustering was performed on the tIC dimensions and an MSM was constructed by optimizing the VAMP2 score af...