Orientation and mode of lipid-binding interaction of human apolipoprotein E C-terminal domain.
ApoE (apolipoprotein E) is an anti-atherogenic lipid transport protein that plays an integral role in lipoprotein metabolism and cholesterol homoeostasis. Lipid association educes critical functional features of apoE, mediating reduction in plasma and cellular cholesterol levels. The 10-kDa CT (C-terminal) domain of apoE facilitates helix-helix interactions in lipid-free state to promote apoE self-association and helix-lipid interactions during binding with lipoproteins, although the mode of lipid-binding interaction is not well understood. We investigated the mode of lipid-binding interaction and orientation of apoE CT domain on reconstituted lipoproteins. Isolated recombinant human apoE CT domain (residues 201-299) possesses a strong ability to interact with phospholipid vesicles, yielding lipoprotein particles with an apparent molecular mass of approximately 600 kDa, while retaining the overall alpha-helical content. Electron microscopy and non-denaturing PAGE analysis of DMPC (dimyristoylphosphatidylcholine)--apoE CT domain lipoprotein complexes revealed discoidal complexes with a diameter of approx. 17 nm. Cross-linking apoE CT domain on discoidal particles yielded dimeric species as the major product. Attenuated total reflectance Fourier transform IR spectroscopy of phospholipid-apoE CT domain complexes reveals that the helical axis is oriented perpendicular to fatty acyl chains of the phospholipid. Fluorescence quenching analysis of DMPC-apoE CT domain discoidal complexes by spin-labelled stearic acid indicated a relatively superficial location of the native tryptophan residues with respect to the plane of the phospholipid bilayer. Taken together, we propose that apoE CT domain interacts with phospholipid vesicles, forming a long extended helix that circumscribes the discoidal bilayer lipoprotein complex.
Raussens
Vincent
V
Structure and Function of Biological Membranes, Université Libre de Bruxelles, CP-206/2, bd. Du Triomphe, B-1050 Brussels, Belgium.
Drury
Jessica
J
Forte
Trudy M
TM
Choy
Nicole
N
Goormaghtigh
Erik
E
Ruysschaert
Jean-Marie
JM
Narayanaswami
Vasanthy
V
D001057:Apolipoproteins E / Q000737:chemistry*; D003432:Cross-Linking Reagents; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D001057:Apolipoproteins E; D003432:Cross-Linking Reagents; D008055:Lipids