Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nam. 2., 166 10 Prague, Czech Republic.
Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668 Warsaw, Poland.
Cells. 2023 Jul 31;12(15):1974. doi: 10.3390/cells12151974.
ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8 (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.
ORPs 是属于氧化固醇结合蛋白家族的脂质转运蛋白。它们促进不同的细胞内膜之间的脂质转移，例如内质网和质膜。我们已经解决了 ORP8 脂质转运结构域（ORD8）的晶体结构。ORD8 表现出由反平行 β-链组成的 β-桶折叠，其中三个 α-螺旋取代了一侧的 β-链。这种混合的 α-β 结构与先前解决的 ORP2 和 ORP3 结构一致。在桶内鉴定出一个大空腔（≈1860Å）作为脂质结合位点。尽管我们无法获得结合脂质的结构，但我们使用基于我们的晶体结构的计算机模拟将 PS 和 PI4P 分子对接入 ORD8 的假定脂质结合位点。短 ORD8（用于结晶学）和全长 ORD8（含盖）之间的比较实验揭示了盖对于稳定脂质结合的重要性。荧光分析揭示了 PS 和 PI4P 的不同转运效率，盖减慢了转运并稳定了货物。粗粒度模拟突出了表面暴露区域和促进脂质双层插入的疏水相互作用。这些发现增强了我们对 ORD8 的理解，包括其结构和脂质转运机制，并为潜在抑制剂的设计提供了结构基础。
