Liu Hang, Tse Chun Mong, Dang Shangyu
Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
The Hong Kong University of Science and Technology-Shenzhen Research Institute, Nanshan, Shenzhen 518057, China.
Proc Natl Acad Sci U S A. 2025 Sep 9;122(36):e2423407122. doi: 10.1073/pnas.2423407122. Epub 2025 Sep 3.
Membrane proteins play crucial roles in numerous biological processes and are important drug targets. However, structural studies of membrane proteins often rely on solubilization with detergents, which may not accurately reflect their native states in a cellular context. Additionally, identifying suitable detergents for individual membrane proteins can be a detailed and time-consuming process. Here, we developed a vesicle-based method that preserves the native lipid environment for subsequent structural and functional studies. Using the bacterial multidrug efflux transporter AcrB as an example, we isolated AcrB-containing vesicles and determined its cryo-EM structure with all protomers in a loose (L) state at 3.88 Å by incorporating our micrograph-based sorting strategy. Notably, compared to the L-state AcrB in liposomes and nanoparticles, the exterior transmembrane helices (TMs) in our map exhibited superior quality, featuring a continuous and clear representation of lα, which is positioned horizontally within the lipid bilayer. We further expanded our method by identifying endogenous membrane proteins, including F-ATPase and respiratory complexes, in vesicles generated using mitochondria from pig hearts. The high-resolution structure of respiratory complex III in vesicles revealed a shared subunit nine between two monomers. Briefly, our method presents a promising and straightforward approach for studying the structure and function of membrane proteins in their native environment, eliminating the need for detergent screening and protein purification.
膜蛋白在众多生物过程中发挥着关键作用，并且是重要的药物靶点。然而，膜蛋白的结构研究通常依赖于用去污剂增溶，这可能无法准确反映它们在细胞环境中的天然状态。此外，为单个膜蛋白鉴定合适的去污剂可能是一个细致且耗时的过程。在此，我们开发了一种基于囊泡的方法，该方法能保留天然脂质环境，用于后续的结构和功能研究。以细菌多药外排转运蛋白AcrB为例，我们分离了含AcrB的囊泡，并通过纳入基于显微照片的分选策略，以3.88 Å的分辨率确定了其处于松散（L）状态的所有原聚体的冷冻电镜结构。值得注意的是，与脂质体和纳米颗粒中的L态AcrB相比，我们图谱中的外部跨膜螺旋（TMs）展现出更高的质量，其lα呈现出连续且清晰的表征，lα在脂质双层中水平定位。我们通过在使用猪心脏线粒体产生的囊泡中鉴定内源性膜蛋白（包括F - ATP酶和呼吸复合体）进一步扩展了我们的方法。囊泡中呼吸复合体III的高分辨率结构揭示了两个单体之间共享的亚基九。简而言之，我们的方法为在天然环境中研究膜蛋白的结构和功能提供了一种有前景且直接的方法，无需进行去污剂筛选和蛋白质纯化。
