Direct Visualization of ATP-Binding Cassette Protein A1 Mediated Nascent High-Density Lipoprotein Biogenesis by High-Speed Atomic Force Microscopy

The ATP-binding cassette protein A1 (ABCA1) is crucial for high-density lipoprotein (HDL) formation, maintaining cholesterol homeostasis. However, the mechanism of ABCA1-mediated HDL formation remains unclear. Here, we reconstituted this process and directly visualized it using high-speed atomic force microscopy by employing a novel linker-based immobilization strategy for side-view imaging of ABCA1 nanodiscs. Our data revealed an approximately 1.6-fold volumetric expansion of the ABCA1 extracellular domain (ECD), coupled with a contraction of the ABCA1-embedded nanodisc, suggesting ATP-dependent transfer of over 300 lipids to the ECD for transient storage. Subsequent addition of apolipoprotein A-I, a major HDL protein, allowed ABCA1 to facilitate nascent HDL particle formation, as it does at the cell membrane. Notably, this study provides the first direct observation of a single ABC protein executing its unique functional cycle with submolecular spatial and subsecond temporal resolution, offering critical insights into "HDL biogenesis", with potential implications for ABCA1-targeted therapeutics for dyslipidemia.