Conformational reorganization of the four-helix bundle of human apolipoprotein E in binding to phospholipid.

Conformational reorganization of the amino-terminal four-helix bundle (22-kDa fragment) of apolipoprotein E (apoE) in binding to the phospholipid dimyristoylphosphatidylcholine (DMPC) to form discoidal particles was investigated by introducing single, double, and triple interhelical disulfide bonds to restrict the opening of the bundle. Interaction of apoE with DMPC was assessed by vesicle disruption, turbidimetric clearing, and gel filtration assays. The results indicate that the formation of apoE.DMPC discoidal particles occurs in a series of steps. A triple disulfide mutant, in which all four helices were tethered, did not form complexes but could release encapsulated 5-(6)-carboxylfluorescein from DMPC vesicles, indicating that the initial interaction does not involve major reorganization of the helical bundle. Initial interaction is followed by the opening of the four-helix bundle to expose the hydrophobic faces of the amphipathic helices. In this step, helices 1 and 2 and helices 3 and 4 preferentially remain paired, since these disulfide-linked mutants bound to DMPC in a manner similar to that of the 22-kDa fragment of apoE4. In contrast, mutants in which helices 2 and 3 and/or helices 1 and 4 paired bound poorly to DMPC. However, all single and double helical pairings resulted in the formation of larger discs than were formed by the 22-kDa fragment, indicating that further reorganization of the helices occurs following the initial opening of the four-helix bundle in which the protein assumes its final lipid-bound conformation. In support of this rearrangement, reducing the disulfide bonds converted the large disulfide mutant discs to normal size.