Bansal Prateek D, Kinnebrew Maia, Rohatgi Rajat, Shukla Diwakar
Department of Chemical and Biomolecular Engineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States.
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, United States.
bioRxiv. 2025 Mar 7:2024.11.25.625241. doi: 10.1101/2024.11.25.625241.
Smoothened (SMO), a member of the G Protein-Coupled Receptor superfamily, mediates Hedgehog signaling and is linked to cancer and birth defects. SMO responds to accessible cholesterol in the ciliary membrane, translocating it via a longitudinal tunnel to its extracellular domain. Reaching a complete mechanistic understanding of the cholesterol translocation process would help in the development of cancer therapies. Experimental data suggests two modes of translocation to support entry of cholesterol from outer and inner membrane leaflets, but the exact mechanism of translocation remains unclear. Using atomistic molecular dynamics simulations (~2 millisecond simulations) and biochemical assays of SMO mutants, we assess the energetic feasibilities of the two modes. We show that the highest energetic barrier for cholesterol translocation from the outer leaflet is lower than that from the inner leaflet. Mutagenesis experiments and complementary simulations of SMO mutants validate the role of critical amino acid residues along the translocation pathways. Our data suggests that cholesterol can take either pathway to enter SMO, thus explaining experimental observations in literature. Thus, our results illuminate the energetics and provide a first molecular description of cholesterol translocation in SMO.
平滑受体（SMO）是G蛋白偶联受体超家族的成员，介导刺猬信号通路，与癌症和出生缺陷有关。SMO对纤毛膜中可及的胆固醇作出反应，通过一条纵向通道将其转运到细胞外结构域。对胆固醇转运过程达成完整的机制理解将有助于癌症治疗的发展。实验数据表明有两种转运模式来支持胆固醇从外膜小叶和内膜小叶进入，但确切的转运机制仍不清楚。我们使用原子分子动力学模拟（约2毫秒模拟）和SMO突变体的生化分析，评估这两种模式的能量可行性。我们表明，胆固醇从外小叶转运的最高能量屏障低于从内小叶转运的能量屏障。SMO突变体的诱变实验和补充模拟验证了沿转运途径的关键氨基酸残基的作用。我们的数据表明胆固醇可以通过任一途径进入SMO，从而解释了文献中的实验观察结果。因此，我们的结果阐明了能量学，并首次对SMO中的胆固醇转运进行了分子描述。
