Studying noncovalent or covalent bond problem between smoothened and cholesterol by molecular dynamics simulation and Markov state model.

Smoothened (SMO) is an attractive therapeutic target for the treatment and prevention of several malignant tumors of the nervous system. The crystal structure of SMO shows cholesterol interacts with residue Asp95 via the noncovalent bond. However, some studies indicate that cholesterol covalently binds to residue Asp95 of SMO. To study these contradictory results, we performed molecular dynamics (MD) simulations and Markov state model (MSM) on SMO in complex with noncovalent-bound and covalent-bound cholesterol. The MD simulated results showed that the noncovalent-bound cholesterol was extremely unstable around the position of residue Asp95 of SMO, while the covalent-bound cholesterol could keep the stable connection with residue Asp95 of SMO. The free energy landscape showed that noncovalent-bound cholesterol had more deep energy wells than covalent-bound cholesterol when it dynamically interacted with the extracellular domain of SMO crystal structure. The MSM results showed the noncovalent-bound cholesterol had more dynamic configuration transformation pathways than the covalent-bound cholesterol. These results theoretically revealed cholesterol should have a covalent bond with residue Asp95 if cholesterol could be stable in the near position of residue Asp95 of SMO. Our studies not only elucidate the covalent binding contradictory issue between cholesterol and residue Asp95 of SMO, but also supply helpful information for antagonists design of SMO.