Structure of the Human Lipid Exporter ABCA1.

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1 Å for the overall structure and 3.9 Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible "lateral access" mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

Rosmarinic Acid Increases Macrophage Cholesterol Efflux through Regulation of ABCA1 and ABCG1 in Different Mechanisms.

Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.

Ene-yne Hydroquinones from a Marine-derived Strain of the Fungus Pestalotiopsis neglecta with Effects on Liver X Receptor Alpha.

Seven unusual new ene-yne hydroquinones (1-3, 5-8), including three rare glycosylated derivatives named pestalotioquinosides A-C (6-8), were obtained from the marine-derived strain SCSIO41403 of the fungus Pestalotiopsis neglecta. Their structures including absolute configurations were elucidated by spectroscopic analysis and induced electronic circular dichroism experiments. In silico molecular docking and in vitro surface plasmon resonance studies showed that pestalotioquinoside C (8) could act as a liver X receptor alpha (LXRα) modulator. Further study showed that LXR target gene ABCA1 was significantly upregulated by 8, which revealed 8 as a potential LXRα agonist.

C-terminal of ABCA1 separately regulates cholesterol floppase activity and cholesterol efflux activity.

ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for cholesterol homeostasis. We recently reported that ABCA1 not only exports excess cholesterol in an apoA-I dependent manner, but that it also flops cholesterol from the inner to the outer leaflet of the plasma membrane. However, the relationship between these two activities of ABCA1 is still unclear. In this study, we analyzed the subcellular localization of ABCA1 by using a newly generated monoclonal antibody against its extracellular domain and the functions of eleven chimera proteins, in which the C-terminal domain of ABCA1 was replaced with those of the other ABCA subfamily members. We identified two motifs important for the functions of ABCA1. Three periodically repeated leucine residues were necessary for the cholesterol floppase activity but not the cholesterol efflux activity, while a VFVNFA motif was essential for both activities of ABCA1. These results suggest that the C-terminal of ABCA1 separately regulates the cholesterol floppase activity and the cholesterol efflux activity.

Computational SNP Analysis and Molecular Simulation Revealed the Most Deleterious Missense Variants in the NBD1 Domain of Human ABCA1 Transporter.

The ATP-binding cassette transporter A1 (ABCA1) is a membrane-bound exporter protein involved in regulating serum HDL level by exporting cholesterol and phospholipids to load up in lipid-poor ApoA-I and ApoE, which allows the formation of nascent HDL. Mutations in the ABCA1 gene, when presents in both alleles, disrupt the canonical function of ABCA1, which associates with many disorders related to lipid transport. Although many studies have reported the phenotypic effects of a large number of ABCA1 variants, the pathological effect of non-synonymous polymorphisms (nsSNPs) in ABCA1 remains elusive. Therefore, aiming at exploring the structural and functional consequences of nsSNPs in ABCA1, in this study, we employed an integrated computational approach consisting of nine well-known in silico tools to identify damaging SNPs and molecular dynamics (MD) simulation to get insights into the magnitudes of the damaging effects. In silico tools revealed four nsSNPs as being most deleterious, where the two SNPs (G1050V and S1067C) are identified as the highly conserved and functional disrupting mutations located in the NBD1 domain. MD simulation suggested that both SNPs, G1050V and S1067C, changed the overall structural flexibility and dynamics of NBD1, and induced substantial alteration in the structural organization of ATP binding site. Taken together, these findings direct future studies to get more insights into the role of these variants in the loss of the ABCA1 function.

N-terminal mutation of apoA-I and interaction with ABCA1 reveal mechanisms of nascent HDL biogenesis.

ApoA-I and ABCA1 play important roles in nascent HDL (nHDL) biogenesis, the first step in the pathway of reverse cholesterol transport that protects against cardiovascular disease. On the basis of the crystal structure of a C-terminally truncated form of apoA-I[Δ(185-243)] determined in our laboratory, we hypothesized that opening the N-terminal helix bundle would facilitate lipid binding. To that end, we structurally designed a mutant (L38G/K40G) to destabilize the N-terminal helical bundle at the first hinge region. Conformational characterization of this mutant in solution revealed minimally reduced α-helical content, a less-compact overall structure, and increased lipid-binding ability. In solution-binding studies, apoA-I and purified ABCA1 also showed direct binding between them. In ABCA1-transfected HEK293 cells, L38G/K40G had a significantly enhanced ability to form nHDL, which suggests that a destabilized N-terminal bundle facilitates nHDL formation. The total cholesterol efflux from ABCA1-transfected HEK293 cells was unchanged in mutant versus WT apoA-I, though, which suggests that cholesterol efflux and nHDL particle formation might be uncoupled events. Analysis of the particles in the efflux media revealed a population of apoA-I-free lipid particles along with nHDL. This model improves knowledge of nHDL formation for future research.

Significance of Cholesterol-Binding Motifs in ABCA1, ABCG1, and SR-B1 Structure.

ABCA1, ABCG1 transporters, and SR-B1 receptor are the major proteins involved in cholesterol efflux from cells. We superposed in silico the location of putative cholesterol (Chol)-binding motifs CRAC/CARC and CCM in human ABCA1, ABCG1, and SR-B1 with (1) transmembrane protein topology, (2) a profile of structural order of protein, and (3) with an influence of single amino acid substitutions on protein structure and function. ABCA1, ABCG1, and SR-B1 molecules contain 50, 19, and 13 Chol-binding motifs, respectively, that are localized either in membrane helices, or at membrane-water interface, or in water-exposed protein regions. Arginine residues in motifs that coincide with molecular recognition features within intrinsically disordered regions of the transporters are suggested to be important in cholesterol binding; cholesterol-arginine interaction may result in the induction of local order in protein structure. Chol-binding motifs in membrane helices may immobilize cholesterol, while motifs at membrane-water interface may be involved into the efflux of "active" cholesterol. Cholesterol may interfere with ATP binding in both nucleotide-binding domains of ABCA1 structure. For ABCA1 and ABCG1, but not for SR-B1, the presence of mirror code as a CARC-CRAC vector couple in the C-terminal helices controlling protein-cholesterol interactions in the outer and inner membrane leaflets was evidenced. We propose the role of Chol-binding motifs with different immersion in membrane in transport of different cholesterol pools by ABCA1 and ABCG1.

Apolipoprotein A-I directly interacts with extracellular domain 1 of human ABCA1.

ATP-binding cassette transporter A1 (ABCA1) is critical for the generation of nascent high-density lipoprotein (HDL) and plays important roles in cholesterol homeostasis. ABCA1 has two large extracellular domains (ECDs), which may interact directly with apolipoprotein A-I (apoA-I). However, the molecular mechanisms underlying HDL formation and the importance of ABCA1-apoA-I interactions in HDL formation remain unclear. We investigated the ABCA1-apoA-I interaction in photo-activated crosslinking experiments using sulfo-SBED-labeled apoA-I. ApoA-I bound to cells expressing ABCA1, but not to untransfected cells or cells expressing non-functional ABCA1. Binding was inhibited by sulfo-SBED-labeled apoA-I, and crosslinking of sulfo-SBED-labeled apoA-I with ABCA1 was inhibited by non-labeled apoA-I, suggesting that sulfo-SBED-labeled apoA-I specifically binds and crosslinks with functional ABCA1. Proteolytic digestion of crosslinked ABCA1 revealed that apoA-I bound the N-terminal half of ABCA1, and that the first ECD of ABCA1 is an apoA-I binding site. Abbreviations: ABC: ATP-binding cassette; apoA-I: apolipoprotein A-I; ATP: adenosine triphosphate; CHAPS: 3-(3-cholamidepropyl)dimethylammonio-1- propanesulphonate; DTT: dithiothreitol; ECD: extra cellular domain; EDTA: ethylenediaminetetraacetic acid; GFP: green fluorescent protein; HA: hemagglutinin; HDL: high density lipoprotein; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; sulfo-SBED: (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido] ethyl-1,3'-dithiopropionate; NHS-ester, N-hydroxysuccinimide-ester.

Is ABCA1 a lipid transfer protein?

ABCA1 functions as a lipid transporter because it mediates the transfer of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apoA-I and related proteins present in the extracellular medium. ABCA1 is a membrane PL translocase and its enzymatic activity leads to transfer of PL molecules from the cytoplasmic leaflet to the exofacial leaflet of a cell plasma membrane (PM). The presence of active ABCA1 in the PM promotes binding of apoA-I to the cell surface. About 10% of this bound apoA-I interacts directly with ABCA1 and stabilizes the transporter. Most of the pool of cell surface-associated apoA-I is bound to lipid domains in the PM that are created by the activity of ABCA1. The amphipathic α-helices in apoA-I confer detergent-like properties on the protein enabling it to solubilize PL and FC in these membrane domains to create a heterogeneous population of discoidal nascent HDL particles. This review focuses on current understanding of the structure-function relationships of human ABCA1 and the molecular mechanisms underlying HDL particle production.

Charged Residues in the C-Terminal Domain of Apolipoprotein A-I Modulate Oligomerization.

Charged residues of the C-terminal domain of human apolipoprotein A-I (apoA-I) were targeted by site-directed mutagenesis. A series of mutant proteins was engineered in which lysine residues (Lys 195, 206, 208, 226, 238, and 239) or glutamate residues (Glu 234 and 235) were replaced by glutamine. The amino acid substitutions did not result in changes in secondary structure content or protein stability. Cross-linking and size-exclusion chromatography showed that the mutations resulted in reduced self-association, generating a predominantly monomeric apoA-I when five or six lysine residues were substituted. The rate of phosphatidylcholine vesicle solubilization was enhanced for all variants, with approximately a threefold rate enhancement for apoA-I lacking Lys 206, 208, 238, and 239, or Glu 234 and 235. Single or double mutations did not change the ability to protect lipolyzed low density lipoprotein from aggregation, but variants lacking >4 lysine residues were less effective in preventing lipoprotein aggregation. ApoA-I mediated cellular lipid efflux from wild-type mice macrophage foam cells was decreased for the variant with five lysine mutations. However, this protein was more effective in releasing cellular phosphatidylcholine and sphingomyelin from Abca1-null mice macrophage foam cells. This suggests that the mutations caused changes in the interaction with ABCA1 transporters and that membrane microsolubilization was primarily responsible for lipid efflux in cells lacking ABCA1. Taken together, this study indicates that ionic interactions in the C-terminal domain of apoA-I favor self-association and that monomeric apoA-I is more active in solubilizing phospholipid bilayers.

Identification of a novel lipid binding motif in apolipoprotein B by the analysis of hydrophobic cluster domains.

Apolipoprotein B (apoB) is a large amphipathic protein that is the structural scaffold for the formation of several classes of lipoproteins involved in lipid transport throughout the body. The goal of the present study was to identify specific domains in the apoB sequence that contribute to its lipid binding properties. A sequence analysis algorithm was developed to identify stretches of hydrophobic amino acids devoid of charged amino acids, which are referred to as hydrophobic cluster domains (HCDs). This analysis identified 78 HCDs in apoB with hydrophobic stretches ranging from 6 to 26 residues. Each HCD was analyzed in silico for secondary structure and lipid binding properties, and a subset was synthesized for experimental evaluation. One HCD peptide, B38, showed high affinity binding to both isolated HDL and LDL, and could exchange between lipoproteins. All-atom molecular dynamics simulations indicate that B38 inserts 3.7Å below the phosphate plane of the bilayer. B38 forms an unusual α-helix with a broad hydrophobic face and polar serine and threonine residues on the opposite face. Based on this structure, we hypothesized that B38 could efflux cholesterol from cells. B38 showed a 12-fold greater activity than the 5A peptide, a bihelical Class A amphipathic helix (EC50 of 0.2658 vs. 3.188µM; p<0.0001), in promoting cholesterol efflux from ABCA1 expressing BHK-1 cells. In conclusion, we have identified novel domains within apoB that contribute to its lipid biding properties. Additionally, we have discovered a unique amphipathic helix design for efficient ABCA1-specific cholesterol efflux.

Position 834 in TM6 plays an important role in cholesterol and phosphatidylcholine transport by ABCA1.

ATP-binding cassette protein A1 (ABCA1) plays a key role in eliminating excess cholesterol from peripheral cells by generating nascent high-density lipoprotein (HDL). However, it remains unclear whether both phospholipids and cholesterol are directly loaded onto apolipoprotein A-I (apoA-I) by ABCA1. To identify the amino acid residues of ABCA1 involved in substrate recognition and transport, we applied arginine scan mutagenesis to residues L821-E843 of human ABCA1 and predicted the environment to which each residue is exposed. The relative surface expression of each mutant suggested that residues L821-E843 pass through the plasma membrane as TM6, and the four residues (S826, F830, L834, and V837) of TM6 are exposed to the hydrophilic internal cavity of ABCA1. Furthermore, we showed that L834 is critical for the function of ABCA1.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear.

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II.

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.

Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane.

The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer's disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.

Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling.

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1-43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185-243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (-10 to -28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (-52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.

Liver X Receptor Exerts Anti-Inflammatory Effects in Colonic Epithelial Cells via ABCA1 and Its Expression Is Decreased in Human and Experimental Inflammatory Bowel Disease.

BACKGROUND: Liver X receptor (LXR) exerts anti-inflammatory effects in macrophages. The aim of this study was to explore the expression and function of LXR in the colonic epithelium under inflammatory conditions. METHODS: The expression of LXR was explored by Western blot and immunohistochemistry in colonic biopsies from patients diagnosed with inflammatory bowel disease (IBD) and control patients. In addition, LXR and its target gene expression were analyzed in the colon from interleukin (IL)-10-deficient (IL-10-/-) and wild-type mice. Caco-2 cells were pretreated with the synthetic LXR agonist GW3965 and further challenged with IL-1ß, the expression of IL-8 and chemokine (C-C motif) ligand (CCL)-28 chemokines, the activation of mitogen-activated protein (MAP) kinases, and the nuclear translocation of the p65 subunit of nuclear factor kappa B was evaluated. Glibenclamide was used as an ABCA1 antagonist. RESULTS: We found that LXR expression was downregulated in colonic samples from patients with IBD and IL-10-/- mice. The nuclear positivity of LXR inversely correlated with ulcerative colitis histologic activity. Colonic IL-1ß mRNA levels negatively correlated with both LXRα and LXRß in the colon of IL-10-/- mice, where a decreased mRNA expression of the LXR target genes ABCA1 and FAS was shown. In addition, IL-1ß decreased the expression of the LXR target gene ABCA1 in cultured intestinal epithelial cells. The synthetic LXR agonist GW3965 led to a decreased nuclear positivity of the p65 subunit of nuclear factor kappa B, a phosphorylation ratio of the p44-42 MAP kinase, and the expression of CCL-28 and IL-8 in IL-1ß-stimulated Caco-2 cells. The pharmacological inhibition of ABCA1 increased the phosphorylation of p44-42 after GW3965 treatment and IL-1ß stimulation. CONCLUSIONS: The LXR-ABCA1 pathway exerts anti-inflammatory effects in intestinal epithelial cells and is impaired in the colonic mucosa of patients with IBD and IL-10-/- mice.

Novel Mechanism of Cholesterol Transport by ABCA5 in Macrophages and Its Role in Dyslipidemia.

Cholesterol homeostasis results from a delicate interplay between influx and efflux of free cholesterol primarily mediated by ABCA1. Here we report downregulation of ABCA1 in hyper-cholesterol conditions in macrophages, which might be responsible for compromised reverse cholesterol transport and hyperlipidemia. Surprisingly, this is countered by the upregulation of a lesser known family member ABCA5 to maintain cholesterol efflux. The relative contribution of ABCA1 and ABCA5 toward cholesterol efflux was evaluated and revealed ABCA5 as the primary efflux mediator under high cholesterol load. These observations were correlated to cholesterol load in circulation in vivo, and we observed an inverse expression profile in mice models of atherosclerosis (ApoE-/-) and hyperlipidemia (PPARα-/-) in response to high cholesterol diet. Observations were further validated in human plasma samples. Simulation studies revealed a unique conformation of ABCA5 proposing a favored route for cholesterol loading onto high-density lipoproteins for reverse cholesterol transport. Thus, our study implicates a functional complementation between these two transporters, formulating an efficient strategy to maintain efflux in cholesterol excess conditions in macrophages.

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation.

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

Structural analysis identifies an escape route from the adverse lipogenic effects of liver X receptor ligands.

Liver X receptors (LXRs) are attractive drug targets for cardiovascular disease treatment due to their role in regulating cholesterol homeostasis and immunity. The anti-atherogenic properties of LXRs have prompted development of synthetic ligands, but these cause major adverse effects-such as increased lipogenesis-which are challenging to dissect from their beneficial activities. Here we show that LXR compounds displaying diverse functional responses in animal models induce distinct receptor conformations. Combination of hydrogen/deuterium exchange mass spectrometry and multivariate analysis allowed identification of LXR regions differentially correlating with anti-atherogenic and lipogenic activities of ligands. We show that lipogenic compounds stabilize active states of LXRα and LXRß while the anti-atherogenic expression of the cholesterol transporter ABCA1 is associated with the ligand-induced stabilization of LXRα helix 3. Our data indicates that avoiding ligand interaction with the activation helix 12 while engaging helix 3 may provide directions for development of ligands with improved therapeutic profiles.