PNNL

Abstract

Apolipoprotein A-I (APOA1) oligomerization is essential for high-density lipoprotein (HDL) metabolism. Naturally occurring mutations can disrupt the normal APOA1 folding and selfassociation, leading to cardiovascular disease. The congenital APOA1 variant p.K131del (APOA1K107del) has been associated with multiple pathologies; while its interaction with lipids has hindered a clear understanding of its functional impairment, structural alterations at all levels have been consistently observed across multiple laboratories. In this study, we confirmed that both APOA1K107del and wild-type APOA1 exhibit similar interactions with lipid monolayers by lipid-protein adsorption assays. Using polyacrylamide gel electrophoresis (PAGE) and sizeexclusion chromatography (SEC), we found that lipid-free APOA1K107del formed fewer dimers, trimers, and tetramers, with a higher proportion of monomers compared to wild-type APOA1.We developed a cross-linking mass spectrometry-based method to detect and quantify differential domain interactions. For the first time, we report that a clinically relevant APOA1 variant exhibits reduced oligomerization, driven by tertiary structural disruptions that persist across oligomeric states. Our results suggest that the central region of APOA1K107del is structurally destabilized, impairing coordinated interactions with both the N- and C-termini and leading to non-specific aggregation. These findings support the hypothesis that proper APOA1 self-association is critical for HDL biogenesis. Additionally, our methodological framework offers a powerful approach for the structural interrogation of other clinically relevant APOA1 proteoforms.