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.     

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).     

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).     

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.     

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.     

Molecular belt models for the apolipoprotein A-I Paris and Milano mutations

Models for the binding of the 200-residue carboxy-terminal domain of two mutants of apolipoprotein A-I (apo A-I), apo A-I(R173C)(Milano) and apo A-I(R151C)(Paris), to lipid in discoidal high-density lipoprotein (HDL) particles are presented. In both models, two monomers of the mutant apo A-I molecule bind to lipid in an antiparallel manner, with the long axes of their helical repeats running perpendicular to the normal of the lipid bilayer to form a single disulfide-linked homodimer. The overall structures of the models of these two mutants are very similar, differing only in helix-helix registration. Thus these models are consistent with experimental observations that reconstituted HDL particles containing apo A-I(Milano) and apo A-I(Paris) are very similar in diameter to reconstituted HDL particles containing wild-type apo A-I, and they support the belief that apo A-I binds to lipid in discoidal HDL particles via the belt conformation.     

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.     

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.     

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.     

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.     

Effect of leucine to phenylalanine substitution on the nonpolar face of a class A amphipathic helical peptide on its interaction with lipid: high resolution solution NMR studies of 4F-dimyristoylphosphatidylcholine discoidal complex

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.     

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.     

Thiol-bearing synthetic peptides retain the antioxidant activity of apolipoproteinA-I(Milano)

Apolipoprotein(apo)A-I(Milano) (R173C) and apoA-I(Paris) (R151C) are rare cysteine variants of wild-type (WT) apoA-I that possess novel antioxidant properties on phospholipid surfaces. Yet, the two variants differ in their ability to inhibit lipid peroxidation. In this study, we used synthetic peptides (18mers) to investigate the structural basis for the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris). A peptide (aa 167-R173C-184) based on the amphipathic alpha helix harboring the R173C mutation inhibited superoxide anion-mediated oxidation of phospholipid in a dose-dependent manner, but it failed to directly quench superoxide anions in aqueous solution, indicating that the peptide acted at the level of phospholipid to inhibit lipid peroxidation just like the full-length cysteine variant. Peptide 145-R151C-162 based on the helical segment containing R151C exhibited the same capacity as peptide 167-R173C-184 to inhibit lipid peroxidation. Thus, the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris) was not governed by the primary amino acid sequence of their individual amphipathic alpha helices, rather contextual constraints within the full-length variants set the difference in antioxidant activity. Cysteine-free peptides were weak inhibitors of lipid peroxidation. These results suggest that thiol-bearing helical peptides based on apoA-I(Milano) may be useful to combat inflammatory related diseases.     

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins.These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.     

Inhibition of virus-induced cell fusion by apolipoprotein A-I and its amphipathic peptide analogs.

Apolipoprotein A-I (apoA-I), the major protein component of serum high-density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid-associating, synthetic amphipathic peptides corresponding to residues 1-33 (apoA-I[1-33]) or residues 66-120 (apoA-I[66-120]) of apoA-I, also inhibited HSV-induced cell fusion, whereas a peptide corresponding to residues 8-33 of apoA-I (apoA-I[8-33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide-mediated fusion inhibition. Consistent with this idea, a series of lipid-binding 22-amino-acid-residue-long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA-I (A-I consensus series), or 18-residue-long model amphipathic peptides (18A series), were found to exert variable levels of fusion-inhibitory activity. The extent of fusion-inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix-forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion-inhibitory activity. Also, the presence of positively charged residues at the polar-nonpolar interface was found to correlate with higher fusion-inhibitory activity.     

Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide.lipid discoidal complexes

Class A amphipathic helical peptides have been shown to mimic apolipoprotein A-I, the major protein component of high density lipoproteins and have been shown to inhibit atherosclerosis in several dyslipidemic mouse models. Previously we reported the NMR structure of Ac-18A-NH2, the base-line model class A amphipathic helical peptide in a 50% (v/v) trifluoroethanol-d3/water mixture, a membrane-mimic environment (Mishra, V. K., Palgunachari, M. N., Anantharamaiah, G. M., Jones, M. K., Segrest, J. P., and Krishna, N. R. (2001) Peptides 22, 567-573). The peptide Ac-18A-NH2 forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Because subtle structural changes in the peptide.lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of recombinant peptide.1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes. The peptide adopts a well defined amphipathic alpha helical structure in association with the lipid at a 1:1 peptide:lipid weight ratio. Nuclear Overhauser effect spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. The pattern of observed peptide-lipid nuclear Overhauser effects is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). Based on the results of chemical cross-linking and molecular modeling, we propose that peptide helices are arranged in a head to tail fashion to cover the edge of the disc. This arrangement of peptides is also consistent with the pKa values of the Lys residues determined previously. Taken together, these results provide for the first time a high resolution structural view of the peptide.lipid discoidal complexes formed by a class A amphipathic alpha helical peptide.     

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.     

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.     

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1

Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.     

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.