Conformation and Lipid Binding of the N-Terminal (1−44) Domain of Human Apolipoprotein A-I

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Abstract
Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1−43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44−243, encoded by exon 4. In contrast to residues 44−243, residues 1−43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1−44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1−44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl β-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration (∼0.7% at 25 °C), sodium dodecyl sulfate, an ionic detergent, above its CMC (∼0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an α-helix inducer, α-helical structure was formed in [1−44]apoA-I up to ∼45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1−44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1−44]apoA-I (w/w) ratio, discoidal complexes with composition ∼4:1 (w/w) and ∼100 Å diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1−44]apoA-I has ∼60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G* helix (residues 8−33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, ∼60% of this region folds into α-helix from random coil. The composition of the 100 Å discoidal complex is ∼5 [1−44]apoA-I and ∼150 DMPC molecules per disk. The helix length of 5 [1−44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.