The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study.
The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529, Sean.Davidson@UC.edu
W Sean
WS
Davidson
George M
GM
Hilliard
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013388:Succinimides
D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D008075:Lipoproteins, HDL; D046911:Macromolecular Substances; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate); D008239:Lysine