PT  - JOURNAL ARTICLE
AU  - T. Bertie Ansell
AU  - Robin A. Corey
AU  - Lucrezia Vittoria Viti
AU  - Maia Kinnebrew
AU  - Rajat Rohatgi
AU  - Christian Siebold
AU  - Mark S. P. Sansom
TI  - The Energetics and Ion Coupling of Cholesterol Transport Through Patched1
AID  - 10.1101/2023.02.14.528445
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.02.14.528445
4099  - http://biorxiv.org/content/early/2023/02/15/2023.02.14.528445.short
4100  - http://biorxiv.org/content/early/2023/02/15/2023.02.14.528445.full
AB  - Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol-1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) 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 1-3 Na+ or 2-3 K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.Competing Interest StatementThe authors have declared no competing interest.