Background:The N-terminal fragment of amyloidogenic apoA-I mutants deposits as fibrils by unknown mechanisms. Results: The G26R mutation partially prevents helix formation of the N-terminal fragment upon lipid binding, thereby facilitating ␤-transition and fibril formation. Conclusion: Membrane binding modulates fibril formation of apoA-I through partially destabilized helical conformation. Significance: The results reveal a new pathway for amyloid fibril formation by apoA-I.

A multidisciplinary approach reveals key insights into the principles of collective cell migration, which is involved in fundamental biological processes. The conformational plasticity of a single molecule, JRAB/MICAL-L2, provides “law and order” in collective cell migration.

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipids-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL3 suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL3.

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 ⌬32-40 and ⌬50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to ␤-sheet structures in all deletion variants except for the ⌬14-22 variant, indicating that residues 14-22 are critical for the ␤-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 ⌬50-58 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. Apolipoprotein (apoA-I) 3 is the major structural and functional constituent of plasma high-density lipoprotein (HDL) This work was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant JP17H03979 (to H. S.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S9.

a b s t r a c tThe N-terminal 1-83 residues of apolipoprotein A-I (apoA-I) have a strong propensity to form amyloid fibrils, in which the 46-59 segment was reported to aggregate to form amyloid-like fibrils. In this study, we demonstrated that a fragment peptide comprising the extreme N-terminal 1-43 residues strongly forms amyloid fibrils with a transition to b-sheet-rich structure, and that the G26R point mutation enhances the fibril formation of this segment. Our results suggest that in addition to the 46-59 segment, the extreme N-terminal region plays a crucial role in the development of amyloid fibrils by the N-terminal fragment of amyloidogenic apoA-I variants. Structured summary of protein interactions:apoA-I and apoA-I bind by fluorescence technology (1, 2, 3) apoA-I and apoA-I bind by atomic force microscopy (View interaction) apoA-I and apoA-I bind by dynamic light scattering (1, 2)