Ansell T Bertie, Corey Robin A, Viti Lucrezia Vittoria, Kinnebrew Maia, Rohatgi Rajat, Siebold Christian, Sansom Mark S P
Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK.
Division of Structural Biology, Wellcome Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK.
bioRxiv. 2023 Feb 15:2023.02.14.528445. doi: 10.1101/2023.02.14.528445.
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 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. data are coupled to 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.
patched1（PTCH1）是哺乳动物刺猬信号通路（HH）的主要肿瘤抑制蛋白，与胚胎发育和组织稳态有关。PTCH1通过一种有争议的机制抑制F类G蛋白偶联受体Smo（SMO），该机制涉及调节纤毛膜内可及的胆固醇水平。通过广泛的分子动力学（MD）模拟和自由能计算来评估胆固醇通过PTCH1的转运，我们发现胆固醇输出存在约15-20 kJ/mol的能量屏障。在模拟中，我们在PTCH1跨膜结构域（TMD）中识别出阳离子结合位点，这可能为胆固醇转运提供能量动力。这些数据与PTCH1突变体的生化分析相结合，以探究跨膜运动与PTCH1活性之间的耦合。通过对Dispatched1（DISP1）的互补模拟，我们发现“向内开放”和溶剂“封闭”状态之间的转变伴随着Na诱导的细胞内螺旋段的收缩。因此，我们的研究结果阐明了PTCH1转运机制的能量学和离子耦合化学计量，即1-3个Na或2-3个K与胆固醇输出耦合，并提供了不同转运状态之间转变的首个分子描述。
