The energetics and ion coupling of cholesterol transport through Patched1

Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein–coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between “inward-open” and solvent “occluded” states is accompanied by Na + -induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na + or two to three K + couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.