Multiple modes of cholesterol translocation in the human Smoothened receptor

Smoothened (SMO), a member of the G Protein-Coupled Receptor superfamily, mediates Hedgehog signaling and is linked to cancer and birth defects. SMO responds to accessible cholesterol in the ciliary membrane, translocating it via a longitudinal tunnel to its extracellular domain. Reaching a complete mechanistic understanding of the cholesterol translocation process would help in the development of cancer therapies. Experimental data suggest two modes of translocation to support entry of cholesterol from outer and inner membrane leaflets, but the exact mechanism of translocation remains unclear. Using atomistic molecular dynamics simulations ([~]2 millisecond simulations) and biochemical assays of SMO mutants, we assess the energetic feasibilities of the two modes. We show that the highest energetic barrier for cholesterol translocation from the outer leaflet is lower than that from the inner leaflet. Mutagenesis experiments and complementary simulations of SMO mutants validate the role of critical amino acid residues along the translocation pathways. Our data suggests that cholesterol can take either pathway to enter SMO, thus explaining experimental observations in the literature. Thus, our results illuminate the energetics and provide a first molecular description of cholesterol translocation in SMO.