The Orientation of Helix 4 in Apolipoprotein A-I-containing Reconstituted High Density Lipoproteins

Abstract

The three-dimensional structure of the high density lipoprotein (HDL) component apolipoprotein (apo) A-I and the molecular basis for its protection against coronary artery disease are unknown. In terms of discoidal HDL particles, there has been a debate as to the orientation of the apoA-I ␣-helices around the disc edge. The "picket fence" model states that the ␣-helical repeats, separated by turns, are arranged parallel to the phospholipid acyl chains of the enclosed lipid bilayer. On the other hand, the "belt" model states that the helical segments run perpendicular to the acyl chains. To distinguish between these models, we used nitroxide spin labels present at various depths in the bilayer of reconstituted HDL (rHDL) to measure the position of Trp residues in single Trp mutants of human proapoA-I. Two mutants were studied; the first contained a Trp at position 108, which was located near the center of helix 4. The second contained a Trp at position 115, two turns along the same helix. The picket fence model predicts that these Trp residues should be at different depths in the bilayer, whereas the belt model predicts that they should be at similar depths. Different sized rHDL particles were produced that contained 2, 3, and >4 molecules of proapoA-I per complex. In each case, parallax analysis indicated that Trp-108 and Trp-115 were present at similar depths of about 6 Å from the center of the bilayer, consistent with helix 4 being oriented perpendicular to the acyl chains (in agreement with the belt model). Similar experiments showed that control transmembrane peptides were oriented parallel to the acyl chains in vesicles, demonstrating that the method was capable of distinguishing between the two models. This study provides one of the first experimental measurements of the location of an apoA-I helix with respect to the bilayer edge.

The Spatial Organization of Apolipoprotein A-I on the Edge of Discoidal High Density Lipoprotein Particles

“…In addition, we published a series of studies in which various single tryptophan mutants of apoA-I were analyzed in discoidal HDL particles containing phospholipids with quenching groups at various positions along the acyl chain. The results clearly showed that all eight 22-amino acid helices in apoA-I were oriented perpendicular to the phospholipid acyl chains (12,13).…”

“…Fig. 6 shows a model for apoA-I termed the "Z-belt" (named because of its shape on the edge of the disc), which we proposed to explain the presence of rHDL particles with three molecules on the disc edge (13). The model is attractive from a symmetry standpoint, because all molecules, including a third, can exist in the same conformation with helices in a belt orientation.…”

“…The model preserves the potential for stabilizing salt bridge interactions between the same residues that were proposed for the double belt, although these must occur intramolecularly in the hairpin model. We have proposed a third model termed the "Z" belt orientation (13). This arrangement is similar to the hairpin except that, instead of traversing back along itself, the molecule proceeds in the same direction on the opposing leaflet, giving the potential for interlocking interactions between the molecules.…”

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

“…This was further supported by a more recent study in which Narayanaswami et al (48) examined the ability of spatially defined nitroxide-labeled phosopholipids to quench the fluorescence of unique single tryptophan apoE3 variants in r-HDL. In addition, a large body of evidence indicates that apoA-I in discoidal HDLs adopt conformations consistent with the belt model (49)(50)(51)(52). Since the sequence of apoE is 44% similar to that of apoA-I and has a similar pattern of 11 and 22 amino acid tandem repeat sequences, it is likely that E422k adopts a similar conformation in NLPs.…”

Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function

“…The general features of the double-belt model, particularly the LL5/5 registry of the two chains ( 19 ), have been confi rmed by multiple laboratories using physical chemical methods (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”