Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.     

Helix orientation of the functional domains in apolipoprotein e in discoidal high density lipoprotein particles

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.     

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.     

Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms

Interaction of the amino- and carboxyl-terminal domains in apolipoprotein (apo) E, referred to as domain interaction, is predicted to be more pronounced in apoE4 than in apoE3 and to underlie the association of apoE4 with Alzheimer and cardiovascular diseases. However, direct physical proof for the domain interaction concept is lacking. To address this issue, fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy were used to probe the spatial proximity of the two domains of apoE. Both methods demonstrated that the two domains are closer in both lipid-free and phospholipid-bound apoE4 than in apoE3 as a result of domain interaction. In addition, as shown by electron paramagnetic resonance, the domains of apoE4 move apart to resemble more closely the distance in apoE3 when the isoforms are bound to triglyceride-rich emulsion particles. These results demonstrate that domain interaction is a structural property of apoE4 and that apoE adopts different conformations when complexed to different lipids.     

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 (1)H,(13)C 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.     

Replacement of helix 1' enhances the lipid binding activity of apoE3 N-terminal domain

The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connecting helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG, introducing a beta-turn. Recombinant helix-to-turn (HT) variant apoE3-NT was produced in Escherichia coli, isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3-NT vs. 2.5 M for wild-type apoE3-NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3-NT was more effective than wild-type apoE3-NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. In lipoprotein binding assays, HT apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent that wild-type apoE3-NT. The results indicate that a mutation at one end of the apoE3-NT four-helix bundle markedly enhances the lipid binding activity of this protein. In the context of lipoprotein associated full-length apoE, increased lipid binding affinity of the N-terminal domain may alter the balance between receptor-active and -inactive conformational states.     

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 alpha-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 alpha-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 alpha-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.     

Fluorescence study of domain structure and lipid interaction of human apolipoproteins E3 and E4

Human apolipoprotein E (apoE) isoforms exhibit different conformational stabilities and lipid-binding properties that give rise to altered cholesterol metabolism among the isoforms. Using Trp-substituted mutations and site-directed fluorescence labeling, we made a comprehensive comparison of the conformational organization of the N- and C-terminal domains and lipid interactions between the apoE3 and apoE4 isoforms. Trp fluorescence measurements for selectively Trp-substituted variants of apoE isoforms demonstrated that apoE4 adopts less stable conformations in both the N- and C-terminal domains compared to apoE3. Consistent with this, the conformational reorganization of the N-terminal helix bundle occurs at lower guanidine hydrochloride concentration in apoE4 than in apoE3 as monitored by fluorescence resonance energy transfer (FRET) from Trp residues to acrylodan attached at the N-terminal helix. Upon binding of apoE3 and apoE4 variants to egg phosphatidylcholine small unilamellar vesicles, similar changes in Trp fluorescence or FRET efficiency were observed for the isoforms, indicating that the opening of the N-terminal helix bundle occurs similarly in apoE3 and apoE4. Introduction of mutations into the C-terminal domain of the apoE isoforms to prevent self-association and maintain the monomeric state resulted in great increase in the rate of binding of the C-terminal helices to a lipid surface. Overall, our results demonstrate that the different conformational organizations of the N- and C-terminal domains have a minor effect on the steady-state lipid-binding behavior of apoE3 and apoE4: rather, self-association property is a critical determinant in the kinetics of lipid binding through the C-terminal helices of apoE isoforms.     

A Unified Scheme for Initiation and Conformational Adaptation of Human Apolipoprotein E N-terminal Domain upon Lipoprotein Binding and for Receptor Binding Activity

We report here a high-resolution NMR structure of the complete receptor-binding domain of human apolipoprotein E3 (apoE3-NT). Similar to the crystal structure of apoE-NT, the NMR structure displayed an elongated four-helix bundle. However, additional unique structural features were also observed. The segments in the N and C termini, which were missing in the crystal structure, formed α-helices having extensive tertiary contacts with the bundle, which oriented these short helices at specific positions for receptor binding activity. Several buried hydrophilic residues observed in the bundle were located strategically between helices 1 and 2 and between helices 3 and 4, significantly destabilizing these helix-helix interfaces. In addition, these buried hydrophilic residues formed buried H-bonds, which may play a key role in specific lipid-free helix bundle recovery. A short helix, nHelix C, was fully solvent-exposed and nearly perpendicular to the bundle. This short helix likely plays a critical role in initiating protein-lipid interaction, causing a preferred conformational adaptation of the bundle at the weaker helix-helix interfaces. This produces an open conformation with two lobes of helices, helices 1 and 4 and helices 2 and 3, which may be the competent conformation for receptor binding activity. Thus, the NMR structure suggests a unified scheme for the initiation and helix bundle opening of apoE-NT upon lipoprotein-binding and for receptor binding activity.Human apolipoprotein E (apoE)² is a 299-residue plasma-exchangeable apolipoprotein with the primary function of transporting lipids from one tissue to another. ApoE performs its functions via interactions with the low-density lipoprotein receptor (LDLR) superfamily (1). The high affinity binding of apoE to the receptors allows apoE-associated lipoprotein particles to be targeted for endocytosis and intracellular degradation. As a subclass of high-density lipoprotein, apoE also influences both cholesterol efflux and influx, thus playing an important role in reverse cholesterol transport (2, 3). Three major isoforms of apoE have been identified: ApoE3 has a cysteine at position 112 and an arginine at position 158, whereas apoE2 has cysteines and apoE4 has arginines at both positions. Although these isoforms differ in only two residues, they show profound functional differences. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer disease, cognitive functioning, immunoregulation, cell signaling, and infectious diseases (4).ApoE is a two-domain protein that contains a 22-kDa N-terminal domain (residues 1-191) and a 10-kDa C-terminal domain (residues 216-299) linked by a protease sensitive hinge region. Although the N-terminal domain of apoE (apoE-NT) is primarily responsible for LDL-receptor binding, the C-terminal domain (apoE-CT) binds to lipoprotein with a high affinity (1). The x-ray crystal structure of lipid-free apoE-NT reveals a globular up-and-down four-helix bundle (5). The major receptor-binding region, residues 130-150, is located on the fourth helix. The positively charged residues (Lys and Arg) in this region are critical for interacting with the negatively charged residues in the receptor (1, 6). This structure only contains residues 24-164, whereas the rest of the regions are disordered. However, experimental evidence indicates that regions beyond residues 24-164 are also critical for LDLR binding activity. For example, deletion of residues 167-185 reduces the apoE3 LDLR binding activity to 15%, and a mutation at position Arg-172 reduces the LDLR binding activity to only ∼2% (7). In addition, an E3K mutant of apoE3 enhances the LDLR binding activity by 2-fold (8). Although the x-ray crystal structure of apoE-NT provides a structural explanation of the major receptor-binding domain of apoE, this structure does not explain the above described important experimental data. Thus, our understanding of the structural basis of the receptor binding activity of apoE remains incomplete.Previous studies using truncation mutants have shown that apoE(1-183) displays nearly 100% LDLR binding activity (9), suggesting that residues beyond position 183 are not important in LDLR binding. We report here a high-resolution NMR structure of the complete LDLR-binding domain of apoE3. Interestingly, our NMR structure shows that the N and C termini form α-helical structures that have extensive contacts with the helix bundle, orienting the two termini at specific positions for potential receptor binding. The NMR structure also displays several novel structural features that may provide the structural basis of a unified scheme for initiation and conformational adaptation of apoE-NT upon lipoprotein binding.     

Apolipoprotein E-low density lipoprotein receptor binding: study of protein-protein interaction in rationally selected docked complexes

Apolipoprotein E (apoE) is an important protein involved in lipid metabolism due to its interaction with members of the low-density lipoprotein receptor (LDLR) family. To further understand the molecular basis for this receptor-binding activity, an apoE fragment containing the receptor binding region (residues 135-151) was docked onto the fifth LDLR ligand binding repeat (LR5) by computational methods. A subset of structures generated by the docking was rationally selected on the grounds of experimental data combined with modeling and was used for further analysis. The application and comparison of two different experimental structures for the apoE fragment underlines the local structural changes occurring in apoE when switching from a receptor-inactive to a receptor-active conformation. The body of interactions occurring at the interface between the two proteins is in very good agreement with the biochemical data available for both apoE and LDLR. Charged residues are involved in numerous ionic interactions and might therefore be important for the specificity of the interaction between apoE and LR5. In addition, the interface also features a tryptophan and a stacking of histidine residues, revealing that the association between the two proteins is not entirely governed by ionic interactions. In particular, the presence of histidine residues in the interface gives a structural basis for the pH-regulated release mechanism of apoE in the endosomes. The proposed molecular basis for apoE binding to LDLR could aid the design of strategies for targeting alterations in lipid transport and metabolism.     

Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins

Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein particles, cofactors for enzymes and ligands for cell-surface receptors. Recent findings with apoA-I and apoE suggest that the tertiary structures of these two members of the human exchangeable apolipoprotein gene family are related. Characteristically, these proteins contain a series of proline-punctuated, 11- or 22-amino acid, amphipathic alpha-helical repeats that can adopt a helix bundle conformation in the lipid-free state. The amino- and carboxyl-terminal regions form separate domains with the latter being primarily responsible for lipid binding. Interaction with lipid induces changes in the conformation of the amino-terminal domain leading to alterations in function; for example, opening of the amino-terminal four-helix bundle in apolipoprotein E upon lipid binding is associated with enhanced receptor-binding activity. The concept of a two-domain structure for the larger exchangeable apolipoproteins is providing new molecular insights into how these apolipoproteins interact with lipids and other proteins, such as receptors. The ways in which structural changes induced by lipid interaction modulate the functionality of these apolipoproteins are reviewed.     

The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger

Exchangeable apolipoproteins can convert between lipid-free and lipid-associated states. The C-terminal domain of human apolipoprotein A-I (apoA-I) plays a role in both lipid binding and self-association. Site-directed spin-label electron paramagnetic resonance spectroscopy was used to examine the structure of the apoA-I C terminus in lipid-free and lipid-associated states. Nitroxide spin-labels positioned at defined locations throughout the C terminus were used to define discrete secondary structural elements. Magnetic interactions between probes localized at positions 163, 217 and 226 in singly and doubly labeled apoA-I gave inter- and intramolecular distance information, providing a basis for mapping apoA-I tertiary and quaternary structure. Spectra of apoA-I in reconstituted HDL revealed a lipid-induced transition of defined random coils and beta-strands into alpha-helices. This conformational switch is analogous to triggered events in viral fusion proteins and may serve as a means to overcome the energy barriers of lipid sequestration, a critical step in cholesterol efflux and HDL assembly.     

Anionic phospholipids inhibit apolipoprotein E--low-density lipoprotein receptor interactions

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family. Lipid-free apoE is not recognized by LDLR, yet interaction with lipid confers receptor recognition properties. Although lipid interaction is known to induce a conformational change in apoE, it is not known if the lipid composition of the resulting complex influences binding. Using reconstituted lipoprotein particles of apoE3 N-terminal (NT) domain and dimyristoylphosphatidylcholine (DMPC), maximal LDLR binding was observed at DMPC:apoE3-NT ratios >2.5:1 (w/w). ApoE3-NT lipid particles prepared with egg sphingomyelin were functional as LDLR ligands while complexes formed with the anionic phospholipids dimyristoylphosphatidylglycerol or dimyristoylphosphatidylserine (DMPS) were not. In the case of apoE3-NT, lipid particles comprised of a mixture of DMPC and DMPS, a DMPS concentration dependent inhibition of LDLR binding activity was observed. Thus, in addition to affecting apoE conformational status, the lipid composition of ligand particles can modulate LDLR binding activity.     

C-terminal interactions of apolipoprotein E4 respond to the postprandial state

Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state.     

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.     

Engineering conformational destabilization into mouse apolipoprotein E. A model for a unique property of human apolipoprotein E4

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer and cardiovascular diseases. ApoE4 differs from the two other common isoforms (apoE2 and apoE3) by its lower resistance to denaturation and greater propensity to form partially folded intermediates. As a first step to determine the importance of stability differences in vivo, we reengineered a partially humanized variant of the amino-terminal domain of mouse apoE (T61R mouse apoE) to acquire a destabilized conformation like that of apoE4. For this process, we determined the crystal structure of wild-type mouse apoE, which, like apoE4, forms a four-helix bundle, and identified two structural differences in the turn between helices 2 and 3 and in the middle of helix 3 as potentially destabilizing sites. Introducing mutations G83T and N113G at these sites destabilized the mouse apoE conformation. The mutant mouse apoE more rapidly remodeled phospholipid than T61R mouse apoE, which supports the hypothesis that a destabilized conformation promotes apoE4 lipid binding.     

Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein e4

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).     

Interaction with amyloid beta peptide compromises the lipid binding function of apolipoprotein E

Apolipoprotein (apo) E is an exchangeable apolipoprotein that plays an integral role in cholesterol transport in the plasma and the brain. It is also associated with protein misfolding or amyloid proteopathy of the beta amyloid peptide (Abeta) in Alzheimer's disease (AD) and cerebral amyloid angiopathy. The C-terminal domain (CT) of apoE encompasses two types of amphipathic alpha helices: a class A helix (residues 216-266) and a class G* helix (residues 273-299). This domain also harbors high-affinity lipoprotein binding and apoE self-association sites that possibly overlap. The objective of this study is to examine if the neurotoxic oligomeric Abeta interacts with apoE CT and if this association affects the lipoprotein binding function of recombinant human apoE CT. Site-specific fluorescence labeling of single cysteine-containing apoE CT variants with donor probes were employed to identify the binding of Abeta bearing an acceptor probe by intermolecular fluorescence resonance energy-transfer analysis. A higher efficiency of energy transfer was noted with probes located in the class A helix than with those located in the class G* helix of apoE CT. In addition, incubation of apoE CT with Abeta severely impaired the lipid binding ability and the overall amount of lipid-associated apoE CT. However, when apoE CT is present in a lipid-bound state, Abeta appears to be localized within the lipid milieu of the lipoprotein particle and not associated with any specific segments of the protein. When our data are taken together, they suggest that Abeta association compromises the fundamental lipoprotein binding function of apoE, which may have implications not only in terms of amyloid buildup but also in terms of the accumulation of cholesterol at extracellular sites.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.