Apolipoprotein A-I Adopts a Belt-like Orientation in Reconstituted High Density Lipoproteins

Abstract

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 Å from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.Because of sedentary lifestyles and high fat diets, atherosclerosis remains one of the leading causes of death in the Western world. It has been demonstrated repeatedly that levels of high density lipoprotein (HDL) 1 and its major protein constituent apolipoprotein A-I (apoA-I) are inversely correlated with the incidence of heart disease (1). Apo-AI, a 28-kDa protein made up of 11-and 22-mer amphipathic helices (Ref. 2; reviewed in Ref. 3), is critical to the formation and stability of the HDL particle in circulation. The process of reverse cholesterol transport is thought to remove excess cholesterol from extra-hepatic tissues such as the arterial wall and return it to the liver for processing (4, 5). ApoA-I performs many critical functions in this pathway. In the lipid poor form, it can interact with the cell surface under the control of the ATP-binding cassette A1 transporter (6). Once apoA-I accumulates phospholipids and cholesterol from the cell surface, it likely forms discoidal complexes that interact with enzymes such as lecithin:cholesterol acetyl transferase and cholesterol ester transfer protein to form the spherical HDL particles commonly found in plasma. These are the particles ...

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.…”

Structure of HDL: Particle Subclasses and Molecular Components

“…The belt model picked up more support with methodologically updated IR experiments performed by Koppaka et al (1999) that contradicted the earlier IR studies supporting the picket fence in rHDL discs. Over the next few years, several laboratories reported results that supported the belt orientation using methodologies like fluorescence energy transfer (Li et al 2000) and lipid-based fluorescence quenching (Panagotopulos et al 2001). With the question of helical orientation largely addressed, much of the focus for the first decade of the 2000s centred on determining the spatial relationships between apoA-I molecules on these discs.…”

High Density Lipoprotein: Assembly, Structure, Cargo, and Functions

“…Over the decades, there have been three basic models of the structures that apoA-I/PL discs form, the models are based on two apoA-I molecules surrounding a phospholipid core and can be described as a picket fence, belt, and hairpin [15]. It is now becoming more apparent that the structure of the apoA-I/PL complex is forever changing to form spherical HDL [15,16,25,35]. The most recent consensus has arisen from some elegant studies conducted by Sean Davidson's group showing that apoA-I molecules in plasma HDL adopt a "cage-like structure" and the size of the HDL particle is determined by the twisting compaction of these apoA-I molecules [36].…”