Computational analysis of the alpha-2 domain of apolipoprotein B - 100, a potential triggering factor in LDL aggregation.

Abstract

Atherosclerosis, the major underlying cause of cardiovascular disease, is believed to arise from the accumulation of low-density lipoprotein (LDL) in the arterial subendothelial space, ultimately leading to plaque formation. It is proposed that the accumulation of LDL is linked to its intrinsic aggregation propensity. Although the native LDL is not prone to aggregation, LDL(-), an electronegative LDL characterized in the plasma, has been shown to prime LDL aggregation in a domino-like behavior similar to amyloidogenic proteins. LDL(-) has also been observed to have a misfolded apolipoprotein B-100 (apo B-100), a huge protein consisting of 4563 amino acid residues. As misfolding of proteins is commonly associated with amyloid formation, apo B-100 is therefore being considered as the possible triggering factor in LDL aggregation. Previous computational studies have implicated the α2 domain to be the aggregation-prone region of apo B-100. In this study, the amyloidogenic properties of the α2 domain of apo B-100 were interrogated using both in silico and in vitro techniques. Since the crystal structure of the 570-amino acid α2 domain of apo B-100 is yet to be solved, we used several secondary structure prediction tools to model putative helical regions that make up the α2 domain. The stability of each of the 17 helices thus identified was further probed using molecular dynamics (MD), with the least stable of the helices considered as potentially amyloidogenic. In a 100 ns simulation window, helices k (YFEKLVGFIDDAVK), m (YHQFVDETNDKIREVTQRLNGEIQA), and p (QQELQRYLSLVGQVYS) were the least stable and appeared to transition to β-structures, the hallmark of amyloidogenesis. When the simulation was extended to longer times, only helices k and p formed stable β-sheets that persisted. Analysis of the data indicates that the final β-sheet conformation was stabilized by the π-π stacking interactions between the aromatic rings of Tyr-1 and Phe-8 for helix k and likely π-π stacking contacts between Arg-6 guanidino group and Tyr-15 ring for helix p. Based on the in silico work, we proceeded to synthesize and spectroscopically characterize helices k, m17-25 (QRLNGEIQA), and p. As expected, k and p formed detectable amyloids, with the latter appearing to be substantially more amyloidogenic based on kinetic aggregation assays. Amyloid fibrils formed by p were confirmed using circular dichroism spectroscopy and transmission electron microscopy. Data obtained could be exploited to further investigate the roles of peptides derived from the α2 domain helices of apo B-100 in triggering LDL aggregation. Based on preliminary data, one of the peptides designed based on this work reduced the aggregation of LDL.