A Mass Spectrometric Determination of the Conformation of Dimeric Apolipoprotein A-I in Discoidal High Density Lipoproteins

摘要

Discoidal forms of high density lipoproteins(HDL)are critical intermediates between lipid-poor apolipoprotein A-I(apo A-I),the major protein constituent of HDL,and the mature spherical forms that comprise the bulk of circulating particles.Thus,many studies have focused on understanding apoA-I structure in discs reconstituted in vitro.Recent theoretical and experimental work supports a"belt"model for apo A-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc.However,disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation.To distinguish among these,we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I.After delipidation and tryptic digestion,mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links.The cross-linking pattern strongly suggests a"double-belt"molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition(LL5/5 orientation).The data also suggests the presence of an additional double-belt orientation with a shifted helical registry(LL5/2 orientation).Furthermore,a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5.A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.