Energetics of cholesterol perception and 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. Competing
hypotheses based on available structures 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 proposed hypotheses. We
show that the energetic barriers for cholesterol translocation from either
leaflets are comparable. 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
contradictory experimental observations in literature. Thus, our results
illuminate the energetics and provide a first molecular description of
cholesterol translocation in SMO.