Rotational and hinge dynamics of discoidal high density lipoproteins probed by interchain disulfide bond formation

To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the ‘9th position’ in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the Cα atoms of G129 (in a ‘9th position’) in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins

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 apoA-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.     

Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I

HDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a “presentation tunnel” gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain “sticky.” Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and “zip-locking” apoA-I into its full LL5/5 registration.     

Double belt structure of discoidal high density lipoproteins: molecular basis for size heterogeneity

We recently proposed an all-atom model for apolipoprotein (apo) A-I in discoidal high-density lipoprotein in which two monomers form stacked antiparallel helical rings rotationally aligned by interhelical salt-bridges. The model can be derived a priori from the geometry of a planar bilayer disc that constrains the hydrophobic face of a continuous amphipathic alpha helix in lipid-associated apoA-I to a plane inside of an alpha-helical torus. This constrains each apoA-I monomer to a novel conformation, that of a slightly unwound, curved, planar amphipathic alpha 11/3 helix (three turns per 11 residues). Using non-denaturing gradient gel electrophoresis, we show that dimyristoylphosphocholine discs containing two apoA-I form five distinct particles with maximal Stokes diameters of 98 A (R2-1), 106 A (R2-2), 110 A (R2-3), 114 A (R2-4) and 120 A (R2-5). Further, we show that the Stokes diameters of R2-1 and R2-2 are independent of the N-terminal 43 residues (the flexible domain) of apoA-I, while the flexible domain is necessary and sufficient for the formation of the three larger complexes. On the basis of these results, the conformation of apoA-I on the R2-2 disc can be modeled accurately as an amphipathic helical double belt extending the full length of the lipid-associating domain with N and C-terminal ends in direct contact. The smallest of the discs, R2-1, models as the R2-2 conformation with an antiparallel 15-18 residue pairwise segment of helixes hinged off the disc edge. The conformations of full-length apoA-I on the flexible domain-dependent discs (R2-3, R2-4 and R2-5) model as the R2-2 conformation extended on the disc edge by one, two or three of the 11-residue tandem amphipathic helical repeats (termed G1, G2 and G3), respectively, contained within the flexible domain. Although we consider these results to favor the double belt model, the topographically very similar hairpin-belt model cannot be ruled out entirely.     

Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins

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 A from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.     

The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins

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 alpha-helices around the disc edge. The "picket fence" model states that the alpha-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 A 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.     

Structures of Discoidal High Density Lipoproteins: A COMBINED COMPUTATIONAL-EXPERIMENTAL APPROACH*

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.

Apolipoprotein A-I (apoA-I) is the principal protein of high density lipoprotein particles (HDL). ApoA-I contains a globular N-terminal domain (residues 1-43) and a lipid-binding C-terminal domain (residues 44-243). Here we propose a detailed model for the smallest discoidal HDL, consisting of two apoA-I molecules wrapped beltwise around a small patch of bilayer containing 160 lipid molecules. The C-terminal domain of each monomer is ringlike, a curved, planar amphipathic alpha helix with an average of 3.67 residues per turn, and with the hydrophobic surface curved toward the lipids. We have explored all possible geometries for forming the dimer of stacked rings, subject to the hypothesis that the optimal geometry will maximize intermolecular salt bridge interactions. The resulting model is an antiparallel arrangement with an alignment matching that of the (nonplanar) crystal structure of lipid-free apoA-I.     

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.     

Apolipoprotein A-I assumes a "looped belt" conformation on reconstituted high density lipoprotein

Apolipoprotein A-I (apoA-I) plays a central role in the reverse cholesterol transport pathway; however, the structural basis for its antiatherogenic effects remains poorly understood. Here we employ EPR spectroscopy and fluorescence resonance energy transfer to elucidate the conformation and relative alignment of apoA-I monomers on discoidal (9.4 nm) reconstituted high density lipoprotein (rHDL). EPR spectroscopy provided evidence for an extended helical secondary structure. Position 139 since it was the only residue examined to display a dynamic motional character consistent with a flexible loop structure. The EPR spectra of nitroxide probes at positions 133 and 146 exhibit spin coupling, indicating that these positions are proximal to an apoA-I paired counterpart on the perimeter of rHDL. fluorescence resonance energy transfer studies employing engineered apoA-I variants possessing a single tryptophan (energy donor) and/or a single cysteine (whose thiol moiety was covalently labeled with an extrinsic energy acceptor) provided evidence that paired apoA-I molecules around the perimeter of rHDL align in an extended antiparallel conformation. Taken together with the observation that the EPR spectra of nitroxide probes positioned at intervening sequence positions (134-145) do not exhibit spin coupling, this has led us to propose a "looped belt" model, wherein residues 133-146 comprise a flexible loop segment that confers to apoA-I an intrinsic ability to adapt its structure to accommodate changing particle lipid content. Specifically, in the looped belt model, with the exception of amino acids 134-145, apoA-I aligns with its counterpart in a helix 5-helix 5 registry, centered at position 139.     

Crystal structure of C-terminal truncated apolipoprotein A-I reveals the assembly of high density lipoprotein (HDL) by dimerization

Apolipoprotein A-I (apoA-I) plays important structural and functional roles in plasma high density lipoprotein (HDL) that is responsible for reverse cholesterol transport. However, a molecular understanding of HDL assembly and function remains enigmatic. The 2.2-? crystal structure of Δ(185-243)apoA-I reported here shows that it forms a half-circle dimer. The backbone of the dimer consists of two elongated antiparallel proline-kinked helices (five AB tandem repeats). The N-terminal domain of each molecule forms a four-helix bundle with the helical C-terminal region of the symmetry-related partner. The central region forms a flexible domain with two antiparallel helices connecting the bundles at each end. The two-domain dimer structure based on helical repeats suggests the role of apoA-I in the formation of discoidal HDL particles. Furthermore, the structure suggests the possible interaction with lecithin-cholesterol acyltransferase and may shed light on the molecular details of the effect of the Milano, Paris, and Fin mutations.     

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.     

Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments

The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on the X-ray crystal structure of a large, lipid-free fragment of apoA-I, a "belt model" was recently proposed. In this model, the helices of two antiparallel apoA-I molecules are extended in a circular arrangement and lie perpendicular to the phospholipid acyl chains. To obtain conclusive information on the spatial organization of apoA-I in discoidal HDL, we engineered three separate cysteine mutants of apoA-I (D9C, A124C, A232C) for specific labeling with the fluorescence probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). The labeled apoA-I was reconstituted into well-defined HDL complexes containing two molecules of protein and dipalmitoylphosphatidylcholine, and the complexes were used in three quantitative fluorescence resonance energy transfer (FRET) experiments to determine the distances between two specific sites in an HDL particle. Comparison of the distances measured by FRET (4.7-7.8 nm) with those predicted from the existing models indicated that neither the picket fence nor the belt model can account for the experimental results; rather, a hairpin folding of each apoA-I monomer with most helices perpendicular to the phospholipid acyl chains and a random head-to-tail and head-to-head arrangement of the two apoA-I molecules in the HDL particles are strongly suggested by the distance and lifetime data.     

Conformation and lipid binding of a C-terminal (198-243) peptide of human apolipoprotein A-I

Human apolipoprotein A-I (apoA-I) is the principle apolipoprotein of high-density lipoproteins that are critically involved in reverse cholesterol transport. The intrinsically flexibility of apoA-I has hindered studies of the structural and functional details of the protein. Our strategy is to study peptide models representing different regions of apoA-I. Our previous report on [1-44]apoA-I demonstrated that this N-terminal region is unstructured and folds into approximately 60% alpha-helix with a moderate lipid binding affinity. We now present details of the conformation and lipid interaction of a C-terminal 46-residue peptide, [198-243]apoA-I, encompassing putative helix repeats 10 and 9 and the second half of repeat 8 from the C-terminus of apoA-I. Far-ultraviolet circular dichroism spectra show that [198-243]apoA-I is also unfolded in aqueous solution. However, self-association induces approximately 50% alpha-helix in the peptide. The self-associated peptide exists mainly as a tetramer, as determined by native electrophoresis, cross-linking with glutaraldehyde, and unfolding data from circular dichroism (CD) and differential scanning calorimetry (DSC). In the presence of a number of lipid-mimicking detergents, above their CMC, approximately 60% alpha-helix was induced in the peptide. In contrast, SDS, an anionic lipid-mimicking detergent, induced helical folding in the peptide at a concentration of approximately 0.003% (approximately 100 microM), approximately 70-fold below its typical CMC (0.17-0.23% or 6-8 mM). Both monomeric and tetrameric peptide can solubilize dimyristoylphosphatidylcholine (DMPC) liposomes and fold into approximately 60% alpha-helix. Fractionation by density gradient ultracentrifugation and visualization by negative staining electromicroscopy demonstrated that the peptide binds to DMPC with a high affinity to form at least two sizes of relatively homogeneous discoidal HDL-like particles depending on the initial lipid:peptide ratio. The characteristics (lipid:peptide weight ratio, diameter, and density) of both complexes are similar to those of plasma A-I/DMPC complexes formed under similar conditions: small discoidal complexes (approximately 3:1 weight ratio, approximately 110 A, and approximately 1.10 g/cm3) formed at an initial 1:1 weight ratio and larger discoidal complexes (approximately 4.6:1 weight ratio, approximately 165 A, and approximately 1.085 g/cm3) formed at initial 4:1 weight ratio. The cross-linking data for the peptide on the complexes of two sizes is consistent with the calculated peptide numbers per particle. Compared to the approximately 100 A disk-like complex formed by the N-terminal peptide in which helical structure was insufficient to cover the disk edge by a single belt, the compositions of these two types of complexes formed by the C-terminal peptide are more consistent with a "double belt" model, similar to that proposed for full-length apoA-I. Thus, our data provide direct evidence that this C-terminal region of apoA-I is responsible for the self-association of apoA-I, and this C-terminal peptide model can mimic the interaction with the phospholipid of plasma apoA-I to form two sizes of homogeneous discoidal complexes and thus may be responsible for apoA-I function in the formation and maintenance of HDL subspecies in plasma.     

Conformation and lipid binding of the N-terminal (1-44) domain of human apolipoprotein A-I

Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1-43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44-243, encoded by exon 4. In contrast to residues 44-243, residues 1-43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1-44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1-44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl beta-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration ( approximately 0.7% at 25 degrees C), sodium dodecyl sulfate, an ionic detergent, above its CMC ( approximately 0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an alpha-helix inducer, alpha-helical structure was formed in [1-44]apoA-I up to approximately 45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1-44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1-44]apoA-I (w/w) ratio, discoidal complexes with composition approximately 4:1 (w/w) and approximately 100 A diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1-44]apoA-I has approximately 60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G helix (residues 8-33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, approximately 60% of this region folds into alpha-helix from random coil. The composition of the 100 A discoidal complex is approximately 5 [1-44]apoA-I and approximately 150 DMPC molecules per disk. The helix length of 5 [1-44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.     

Identification and structural ramifications of a hinge domain in apolipoprotein A-I discoidal high-density lipoproteins of different size

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.     

Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.     

Novel changes in discoidal high density lipoprotein morphology: a molecular dynamics study

ApoA-I is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-I. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-I particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-I, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstroms and 78 angstroms by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules.     

Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the Delta(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.