Mechanism of the cholesterol transport in smoothened.

Abstract

Cholesterol (CHL) has been identified as the endogenous agonist of Smoothened (SMO), a Class F G-Protein Coupled Receptor (GPCR). SMO, a member of the Hedgehog signaling pathway, is endogenously inhibited by Patched (PTCH). How PTCH inhibits SMO is unclear - however, the leading hypothesis states that PTCH controls SMO's accessibility to membrane cholesterol by being a putative cholesterol transporter. On PTCH's inhibition by Sonic Hedgehog (ShH), cholesterol can then travel through the upper leaflet in SMO to the sterol binding site in SMO CRD. SMO structures with sterols at different points along the pathway have been observed, but a study connecting the different sterol poses is lacking. Here, we use millisecond-scale atomistic molecular dynamics simulations in tandem with Markov State Modeling (MSM) to probe the cholesterol transport dynamics of SMO. We observe that SMO can indeed transport cholesterol from the membrane to the CRD site, a probable path exists for the same. A maximum barrier of ∼7.5 kcal/mol is observed at the membrane-SMO interface for the entry of cholesterol into the SMO Transmembrane Domain (TMD). This entry involves the rocking motion of cholesterol, which is elucidated by the tail-first entry of cholesterol entry in the TMD. This is followed by translation to the TMD-CRD interface, where CHL might showing flipping motions. The final step is the translation from the TMD-CRD interface to the CRD binding site. We observe that simulations with cholesterol make SMO inherently active, further corroborating the presence of multiple-binding sites of CHL in SMO. Thus, our study gives dynamical insights into a long-standing question in the field of endogenous SMO activation.