Deciphering high density lipoprotein (HDL) structure-function: Detailed analysis of HDL subfractions reveals molecular differences leading to atherosclerosis risk

High-density lipoproteins (HDL) are crucial for cardiovascular health. HDL dysfunction is strongly linked to atherosclerosis and other diseases. Although HDL is often treated as a single entity, it comprises a spectrum of subfractions differing in size, structure, and composition. Understanding the ultrastructure and function of these subfractions is essential for uncovering the molecular mechanisms behind HDL dysfunction and improving disease risk prediction. We employed Small-Angle X-ray Scattering (SAXS) and cryogenic-electron tomography (cryo-ET) to analyze the structural features of total HDL and its subfractions: HDL 2b , HDL 2a , and HDL 3 . We present updated, detailed structural models showing that the structure of HDL changes dramatically from the smallest to the largest subfraction, with HDL 3 partially exposing the core to the particle's surface. Moreover, total HDL reflects the weighted sum of its subfractions, emphasizing that analysis of total HDL alone may be misleading. HDL subfractions were also studied in serum samples from 16 individuals classified as low- or high-risk for atherosclerosis. Even though biochemical differences appeared mainly in HDL 2 , structural differences were most pronounced in HDL 3 addressing that particle composition alone cannot fully distinguish HDL dysfunction. Multimodal analysis integrating structural and biochemical data was able to separate risk groups and revealed correlations between structural parameters and cardiovascular risk status. Our findings support the importance of analyzing HDL subfractions to uncover molecular drivers of disease risk and suggest candidate biomarkers for atherosclerosis upon validation in larger cohorts.