Padmaja Dhanasekaran

Mechanism of ATP-binding Cassette Transporter A1-mediated Cellular Lipid Efflux to Apolipoprotein A-I and Formation of High Density Lipoprotein Particles

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.

Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model

Detailed structural information on human exchangeable apolipoproteins (apo) is required to understand their functions in lipid transport. Using a series of deletion mutants that progressively lacked different regions along the molecule, we probed the structural organization of lipid-free human apoA-I and the role of different domains in lipid binding, making comparisons to apoE, which is a member of the same gene family and known to have two structural domains. Measurements of α-helix content by CD in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that deletion of the amino-terminal or central regions disrupts the tertiary organization, whereas deletion of the carboxyl terminus has no effect on stability and induces a more cooperative structure. These data are consistent with the lipid-free apoA-I molecule being organized into two structural domains similar to apoE; the amino-terminal and central parts form a helix bundle, whereas the carboxyl-terminal α-helices form a separate, less organized structure. The binding of the apoA-I variants to lipid emulsions is modulated by reorganization of the helix bundle structure, because the rate of release of heat on binding is inversely correlated with the stability of the helix bundle. Based on these observations, we propose that there is a two-step mechanism for lipid binding of apoA-I: apoA-I initially binds to a lipid surface through amphipathic α-helices in the carboxyl-terminal domain, followed by opening of the helix bundle in the amino-terminal domain. Because apoE behaves similarly, this mechanism is probably a general feature for lipid interaction of other exchangeable apolipoproteins, such as apoA-IV.

Effects of Apolipoprotein A-I on ATP-binding Cassette Transporter A1-mediated Efflux of Macrophage Phospholipid and Cholesterol: Formation of nascent high density lipoprotein particles

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing ∼3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal α-helix (residues 223–243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20–450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.

Scavenger Receptor Class B Type I-mediated Cholesteryl Ester-selective Uptake and Efflux of Unesterified Cholesterol INFLUENCE OF HIGH DENSITY LIPOPROTEIN SIZE AND STRUCTURE

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (Bmax ∼420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small (∼8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I·SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.

Influence of ApoA-I Structure on the ABCA1-mediated Efflux of Cellular Lipids

The influence of apolipoprotein (apo) A-I structure on ABCA1-mediated efflux of cellular unesterified (free) cholesterol (FC) and phospholipid (PL) is not well understood. To address this issue, we used a series of apoA-I mutants to examine the contributions of various domains in the molecule to ABCA1-mediated FC and PL efflux from mouse J774 macrophages and human skin fibroblasts. Irrespective of the cell type, deletion or disruption of the C-terminal lipid-binding domain of apoA-I drastically reduced the FC and PL efflux (∼90%), indicating that the C-terminal amphipathic α-helix is required for high affinity microsolubilization of FC and PL. Deletion in the N-terminal region of apoA-I also reduced the lipid efflux (∼30%) and increased the Km about 2-fold compared with wild type apoA-I, whereas deletion of the central domain (Δ123–166) had no effect on either Km or Vmax. These results indicate that ABCA1-mediated lipid efflux is relatively insensitive to the organization of the apoA-I N-terminal helix-bundle domain. Alterations in apoA-I structure caused parallel changes in its ability to bind to a PL bilayer and to induce efflux of FC and PL. Overall, these results are consistent with a two-step model for ABCA1-mediated lipid efflux. In the first step, apoA-I binds to ABCA1 and hydrophobic α-helices in the C-terminal domain of apoA-I insert into the region of the perturbed PL bilayer created by the PL transport activity of ABCA1, thereby allowing the second step of lipidation of apoA-I and formation of nascent high density lipoprotein particles to occur.

Effects of polymorphism on the lipid interaction of human apolipoprotein E

ApoE exists as three common isoforms, apoE2, apoE3, and apoE4; apoE2 and apoE3 preferentially bind to high density lipoproteins, whereas apoE4 prefers very low density lipoproteins (VLDL). To understand the molecular basis for the different lipoprotein distributions of these isoforms in human plasma, we examined the lipid-binding properties of the apoE isoforms and some mutants using lipid emulsions. With both large (120 nm) and small (35 nm) emulsion particles, the binding affinity of apoE4 was much higher than that of apoE2 and apoE3, whereas the maximal binding capacities were similar among the three isoforms. The 22-kDa N-terminal fragment of apoE4 displayed a much higher binding capacity than did apoE2 and apoE3. The apoE4(E255A) mutant, which has no electrostatic interaction between Arg61 and Glu255, showed binding behavior similar to that of apoE3, indicating that N- and C-terminal domain interaction in apoE4 is responsible for its high affinity for lipid. In addition, the apoE3(P267A) mutant, which is postulated to contain a long α-helix in the C-terminal domain, had significantly decreased binding capacities for both sizes of emulsion particle, suggesting that the apoE4 preference for VLDL is not due to a stabilized long α-helical structure. Isothermal titration calorimetry measurements showed that there is no significant difference in thermodynamic parameters for emulsion binding among the apoE isoforms. However, fluorescence measurements of 8-anilino-1-naphthalenesulfonic acid binding to apoE indicated that apoE4 has more exposed hydrophobic surface compared with apoE3 mainly due to the different tertiary organization of the C-terminal domain. The less organized structure in the C-terminal domain of apoE4 leads to the higher affinity for lipid, contributing to its preferential association with VLDL. In fact, we found that apoE4 binds to VLDL with higher affinity compared with apoE3.

Effects of Lipid Interaction on the Lysine Microenvironments in Apolipoprotein E

Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1–191) in the lipid-free and lipid-associated states. As shown by 1H,13C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3·dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK a values above 10. In apoE3·DMPC complexes, however, all eight lysines were resolved, and the pK a values were 9.2–11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK a values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3·DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.

Influence of Tertiary Structure Domain Properties on the Functionality of Apolipoprotein A-I

The tertiary structure of apolipoprotein (apo) A-I and the contributions of structural domains to the properties of the protein molecule are not well defined. We used a series of engineered human and mouse apoA-I molecules in a range of physical-biochemical measurements to address this issue. Circular dichroism measurements of alpha-helix thermal unfolding and fluorescence spectroscopy measurements of 8-anilino-1-napthalenesulfonic acid binding indicate that removal of the C-terminal 54 amino acid residues from human and mouse apoA-I has similar effects; the molecules are only slightly destabilized, and there is a decrease in hydrophobic surface exposure. These results are consistent with both human and mouse apoA-I adopting a two-domain tertiary structure, comprising an N-terminal antiparallel helix bundle domain and a separate less ordered C-terminal domain. Mouse apoA-I is significantly less resistant than human apoA-I to thermal and chemical denaturation; the midpoint of thermal unfolding of mouse apoA-I at 45 degrees C is 15 degrees C lower and the midpoint of guanidine hydrochloride denaturation (D1/2) occurs at 0.5 M as compared to 1.0 M for human apoA-I. These differences reflect the overall greater stability of the helix bundle formed by residues 1-189 in human apoA-I. Measurements of the heats of binding to egg phosphatidylcholine (PC) small unilamellar vesicles and the kinetics of solubilization of dimyristoyl PC multilamellar vesicles indicate that the more stable human helix bundle interacts poorly with lipids as compared to the equivalent mouse N-terminal domain. The C-terminal domain of human apoA-I is much more hydrophobic than that of mouse apoA-I; in the lipid-free state the human C-terminal domain (residues 190-243) is partially alpha-helical and undergoes cooperative unfolding (D1/2 = 0.3 M) whereas the isolated mouse C-terminal domain (residues 187-240) is disordered in dilute solution. The human C-terminal domain binds to lipid surfaces much more avidly than the equivalent mouse domain. Human and mouse apoA-I have very different tertiary structure domain contributions for achieving functionality. It is clear that the stability of the N-terminal helix bundle, and the hydrophobicity and alpha-helix content of the C-terminal domain, are critical factors in determining the overall properties of the apoA-I molecule.

Molecular Mechanism of Apolipoprotein E Binding to Lipoprotein Particles

The exchangeability of apolipoprotein (apo) E between lipoprotein particles such as very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) is critical for lipoprotein metabolism, but despite its importance, the kinetics and mechanism of apoE-lipoprotein interaction are not known. We have used surface plasmon resonance (SPR) to monitor in real time the reversible binding of apoE to human VLDL and HDL(3); biotinylated lipoproteins were immobilized on a streptavidin-coated SPR sensor chip, and solutions containing various human apoE molecules at different concentrations were passed across the surface. Analysis of the resultant sensorgrams indicated that the apoE3-lipoprotein interaction is a two-step process. After an initial interaction, the second slower step involves opening of the N-terminal helix bundle domain of the apoE molecule. Destabilization of this domain leads to more rapid interfacial rearrangement which is seen when the lipoprotein binding of apoE4 is compared to that of apoE3. The resultant differences in interfacial packing seem to underlie the differing abilities of apoE4 and apoE3 to bind to VLDL and HDL(3). The measured dissociation constants for apoE binding to these lipoprotein particles are in the micromolar range. C-Terminal truncations of apoE to remove the lipid binding region spanning residues 250-299 reduce the level of binding to both types of lipoprotein, but the effect is weaker with HDL(3); this suggests that protein-protein interactions are important for apoE binding to this lipoprotein while apoE-lipid interactions are more significant for VLDL binding. The two-step mechanism of lipoprotein binding exhibited by apoE is likely to apply to other members of the exchangeable apolipoprotein family.

Conformational flexibility of the N-terminal domain of apolipoprotein a-I bound to spherical lipid particles.

Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal alpha-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the N- and C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229. Neither mutagenesis nor the pyrene labeling caused discernible changes in the lipid-free structure and lipid interaction of apoA-I. Taking advantage of a significant increase in fluorescence when a pyrene-labeled helix is in contact with the lipid surface, we monitored the behaviors of the N- and C-terminal helices upon binding of apoA-I to egg PC small unilamellar vesicles. Comparison of the binding isotherms for pyrene-labeled apoA-I as well as a C-terminal helical peptide suggests that an increase in surface concentration of apoA-I causes dissociation of the N-terminal helix from the surface leaving the C-terminal helix attached. Consistent with this, isothermal titration calorimetry measurements showed that the enthalpy of apoA-I binding to the lipid surface under near saturated conditions is much less exothermic than that for binding at a low surface concentration, indicating the N-terminal helix bundle is out of contact with lipid at high apoA-I surface concentrations. Interestingly, the presence of cholesterol significantly induces the open conformation of the helix bundle. These results provide insight into the multiple lipid-bound conformations that the N-terminal helix bundle of apoA-I can adopt on a lipid or lipoprotein particle, depending upon the availability of space on the surface and the surface composition.