Mechanisms of aggregation and fibril formation of the amyloidogenic N-terminal fragment of apolipoprotein A-I

The N-terminal (1–83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14–22 and 50–58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1–83 fragment (apoA-I 1–83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14–22 completely inhibits fibril formation of apoA-I 1–83/G26R, whereas Δ32–40 and Δ50–58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to β-sheet structures in all deletion variants except for the Δ14–22 variant, indicating that residues 14–22 are critical for the β-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1–83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Δ50–58 variant was entropically unfavorable, indicating that residues 50–58 entropically promote the nucleation step in fibril formation of apoA-I 1–83/G26R. Moreover, a residue-level structural investigation of apoA-I 1–83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.