The Energetics and Ion Coupling of Cholesterol Transport Through Patched1
The Energetics and Ion Coupling of Cholesterol Transport Through Patched1
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DOI：
10.1101/2023.02.14.528445
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发表时间：
2023-02
期刊：
影响因子：
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通讯作者：
T. Ansell;R. Corey;Lucrezia Vittoria Viti;M. Kinnebrew;R. Rohatgi;C. Siebold;M. Sansom
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文献类型：
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作者：
T. Ansell;R. Corey;Lucrezia Vittoria Viti;M. Kinnebrew;R. Rohatgi;C. Siebold;M. Sansom
Patched 1（PTCH 1）是哺乳动物Hedgehog（HH）信号通路的主要肿瘤抑制蛋白，与胚胎发生和组织稳态有关。PTCH 1通过一种有争议的机制抑制F类G蛋白偶联受体Smoothened（SMO），该机制涉及调节睫状膜内的可及胆固醇水平。使用广泛的分子动力学（MD）模拟和自由能计算来评估胆固醇通过PTCH 1的运输，我们发现胆固醇输出的能量势垒为~15-20 kJ mol-1。在模拟中，我们确定的PTCH 1跨膜结构域（TMD）内的阳离子结合位点，这可能会提供胆固醇运输的有力推动。将计算机模拟数据与PTCH 1突变体的体内生物化学测定相结合，以探测跨膜运动与PTCH 1活性之间的偶联。使用Dispatched 1（DISP 1）的互补模拟，我们发现，“向内开放”和溶剂“闭塞”状态之间的过渡是伴随着Na+诱导捏胞内螺旋段。因此，我们的研究结果阐明了PTCH 1运输机制的能量学和离子耦合化学计量学，其中1-3 Na+或2-3 K+耦合到胆固醇出口，并提供了不同运输状态之间的转换的第一个分子描述。
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.