The energetics and ion coupling of cholesterol transport through Patched1

  • Sci Adv. 2023 Aug 25;9(34):eadh1609. doi: 10.1126/sciadv.adh1609.
T Bertie Ansell  1 ,  Robin A Corey  1  2 ,  Lucrezia Vittoria Viti  3 ,  Maia Kinnebrew  4 ,  Rajat Rohatgi  4 ,  Christian Siebold  3 ,  Mark S P Sansom  1
Affiliations
  • 1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
  • 2. School of Physiology, Pharmacology and Neuroscience, Bristol University, Bristol BS8 1TD, UK.
  • 3. Division of Structural Biology, Wellcome Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK.
  • 4. Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, CA, USA.
PMID: 37611095 DOI: 10.1126/sciadv.adh1609
Abstract

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.