RT Journal Article
SR Electronic
T1 The Energetics and Ion Coupling of Cholesterol Transport Through Patched1
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.02.14.528445
DO 10.1101/2023.02.14.528445
A1 T. Bertie Ansell
A1 Robin A. Corey
A1 Lucrezia Vittoria Viti
A1 Maia Kinnebrew
A1 Rajat Rohatgi
A1 Christian Siebold
A1 Mark S. P. Sansom
YR 2023
UL http://biorxiv.org/content/early/2023/02/15/2023.02.14.528445.abstract
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.