Lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III

The lipid-interacting properties of the N-terminal domain of human apolipoprotein C-III (apo C-III) were investigated. By molecular modeling, we predicted that the 6-20 fragment of apo C-III is obliquely orientated at the lipid/water interface owing to an asymmetric distribution of the hydrophobic residues when helical. This is characteristic of 'tilted peptides' originally discovered in viral fusion proteins and later in various proteins including some involved in lipoprotein metabolism. Since most tilted peptides were shown to induce liposome fusion in vitro, the fusogenic capacity of the 6-20 fragment of apo C-III was tested on unilamellar liposomes and compared with the well characterized SIV fusion peptide. Mutants were designed by molecular modeling to assess the role of the hydrophobicity gradient in the fusion. FTIR spectroscopy confirmed the predominantly helical conformation of the peptides in TFE solution and also in lipid-peptide complexes. Lipid-mixing experiments showed that the apo C-III (6-20) peptide is able to increase the fluorescence of a lipophilic fluorescent probe. The vesicle fusion was confirmed by core-mixing and leakage assays. The hydrophobicity gradient plays a key role in the fusion process because the mutant with no hydrophobic asymmetry but the same mean hydrophobicity as the wild type does not induce significant lipid fusion. The apo C-III (6-20) fragment is, however, less fusogenic than the SIV peptide, in agreement with their respective mean hydrophobicity. Since lipid fusion should not be the physiological function of the N-terminal domain of apo CIII, we suggest that its peculiar distribution of hydrophobic residues is important for the lipid-binding properties of apo C-III and should be involved in apolipoprotein and lipid exchanges crucial for triglyceride metabolism.     

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

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

Mimicking lipid-binding-induced conformational changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.     

Differences in the binding capacity of human apolipoprotein E3 and E4 to size-fractionated lipid emulsions.

We describe sensitive new approaches for detecting and quantitating protein-lipid interactions using analytical ultracentrifugation and continuous size-distribution analysis [Schuck (2000) Biophys. J.78, 1606-1619]. The new methods were developed to investigate the binding of human apolipoprotein E (apoE) isoforms to size-fractionated lipid emulsions, and demonstrate that apoE3 binds preferentially to small lipid emulsions, whereas apoE4 exhibits a preference for large lipid particles. Although the apparent binding affinity for large emulsions is similar (Kd approximately 0.5 micro m), the maximum binding capacity for apoE4 is significantly higher than for apoE3 (3.0 and 1.8 amino acids per phospholipid, respectively). This indicates that apoE4 has a smaller binding footprint at saturation. We propose that apoE isoforms differentiate between lipid surfaces on the basis of size, and that these differences in lipid binding are due to a greater propensity of apoE4 to adopt a more compact closed conformation. Implications for the role of apoE4 in blood lipid transport and disease are discussed.     

Structural properties and lipid binding of human apolipoprotein A-IV

The in vivo affinity of human apolipoprotein A-IV (apo-A-IV) for plasma lipoproteins is considerably less than that of other apolipoproteins. We have therefore studied its spectroscopic properties and its association with model chylomicrons to investigate its structural characteristics and to define their influence upon its affinity for lipids. Fluorescence emission spectra of apo-A-IV in dilute aqueous solution revealed that its single tryptophan residue resides in a pH-sensitive hydrophobic domain, which is maximally protected from iodide quenching at pH 7.5. Denaturation of apo-A-IV by guanidine hydrochloride caused a multiphasic fluorescence emission red shift, with an unusual enhancement of quantum yield. Circular dichroism spectroscopy of apo-A-IV demonstrated negative ellipticity maxima at 210 and 222 nm, consistent with 54% alpha-helical structure. The alpha-helicity of apo-A-IV as measured by [theta]222 was also pH-sensitive and displayed a distinctive decrease between pH 7.0 and 8.0. Apo-A-IV was exquisitely sensitive to denaturation by guanidine hydrochloride, and its estimated free energy of stabilization in aqueous solution was near zero. Apo-A-IV bound to the surface of Sf greater than 400 particles of a phospholipid-triglyceride emulsion in a noncooperative, concentration-dependent manner. The affinity of apo-A-IV for these model chylomicrons was influenced by changes in pH or addition of guanidine hydrochloride in a manner which correlated well with the structural changes observed under similar conditions. We conclude that human apolipoprotein A-IV possesses several biophysical properties characteristic of the better studied plasma apolipoproteins, yet, apo-A-IV appears to be marginally stable in aqueous solution and its structural characteristics and lipid binding properties are particularly sensitive to environment.     

Self-association and lipid binding properties of the lipoprotein initiating domain of apolipoprotein B

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His6-tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly.     

The receptor-binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments

To identify the domain of apolipoprotein E (apo-E) involved in binding to low density lipoprotein (LDL) receptors on cultured human fibroblasts, apo-E was cleaved and the fragments were tested for receptor binding activity. Two large thrombolytic peptides (residues 1-191 and 216-299) of normal apo-E3 were combined with the phospholipid dimyristoylphosphatidylcholine (DMPC) and tested for their ability to compete with 125I-LDL for binding to the LDL (apo-B,E) receptors on human fibroblasts. The NH2-terminal two-thirds (residues 1-191) of apo-E3 was as active as intact apo-E3 . DMPC, while the smaller peptide (residues 216-299) was devoid of receptor-binding activity. When apo-E3 was digested with cyanogen bromide (CNBr) and the four largest CNBr fragments were combined with DMPC and tested, only one fragment competed with 125I-LDL for binding to cultured human fibroblasts (CNBr II, residues 126-218). This fragment possessed binding activity similar to that of human LDL. The 125I-labeled CNBr II . DMPC complex also demonstrated high affinity, calcium-dependent saturable binding to solubilized bovine adrenal membranes. The binding of CNBr II . DMPC was inhibited by 1,2-cyclohexanedione modification of arginyl residues or diketene modification of lysyl residues. In addition, the CNBr II had to be combined with DMPC before it demonstrated any receptor-binding activity. Pronase treatment of the membranes abolished the ability of this fragment to bind to the apo-B,E receptors. This same basic region in the center of the molecule has been implicated as the apo-B,E receptor-binding domain not only by this study but also by other studies showing that 1) natural mutants of apo-E that display defective binding have single amino acid substitutions at residues 145, 146, or 158; and 2) the apo-E epitope of the monoclonal antibody 1D7, which inhibits apo-E binding, is centered around residues 139-146.     

Interaction of the N-terminal domain of apolipoprotein E4 with heparin

Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Sodium dodecyl sulfate-resistant complexes of Alzheimer's amyloid beta-peptide with the N-terminal, receptor binding domain of apolipoprotein E

Immunocytochemical, biochemical, and molecular genetic studies indicate that apolipoprotein E (apoE) plays an important role in the process of amyloidogenesis-beta. However, there is still no clear translation of these data into the pathogenesis of amyloidosis-beta. Previous studies demonstrated sodium dodecyl sulfate (SDS)-resistant binding of apoE to the main component of Alzheimer's amyloid-A beta and modulation of A beta aggregation by apoE in vitro. To more closely characterize apoE-A beta interactions, we have studied the binding of thrombolytic fragments of apoE3 to A beta in vitro by using SDS-polyacrylamide gel electrophoresis and intrinsic fluorescence quenching. Here we demonstrate that SDS-resistant binding of A beta is mediated by the receptor-binding, N-terminal domain of apoE3. Under native conditions, both the N- and C-terminal domains of apoE3 bind A beta; however, the former does so with higher affinity. We propose that the modulation of A beta binding to the N-terminal domain of apoE is a potential therapeutic target for the treatment of amyloidosis-beta.     

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.     

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.     

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.     

A monomeric human apolipoprotein E carboxyl-terminal domain

ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family. In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation. The aggregation property presents a major difficulty for the structural determination of this protein. A high-level expression system of the apoE C-terminal domain is reported here. Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant. This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E). The solubility of the mutant is significantly increased ( approximately 10-fold). Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL. CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein. DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain. In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins. These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity. Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.     

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.     

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.     

An N-terminal three-helix fragment of the exchangeable insect apolipoprotein apolipophorin III conserves the lipid binding properties of wild-type protein

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.     

Human apolipoprotein E: polymorphism and binding domain of the receptors

This paper summarizes present knowledge of the LDL receptor-binding domain of apolipoprotein E, with special emphasis on the influence of apolipoprotein polymorphism on the interaction with apo B/E receptors.     

Lipid association-induced N- and C-terminal domain reorganization in human apolipoprotein E3

Apolipoprotein E (apoE) is a 299 amino acid, anti-atherogenic protein that plays a key role in regulating plasma lipoprotein metabolism. It is composed of an N-terminal (NT) domain (residues 1-191) that is responsible for binding to members of the low density lipoprotein receptor family and a C-terminal (CT) domain (residues 216-299) that anchors the protein to lipoprotein particles by virtue of its high-affinity lipid binding characteristics. Isoform-specific differences in the NT domain that modulate the lipoprotein binding preference elicited by the CT domain suggest the existence and importance of domain interactions in this protein. Employing steady state fluorescence quenching and resonance energy transfer techniques, spatial proximity relationships between the N- and C-terminal domains were investigated in recombinant human apoE3. ApoE3 containing a single Trp at position 264 and an N-iodoacetyl-N'-(5-sulfo-1-napthyl) ethylenediamine (AEDANS) moiety covalently attached to the lone Cys residue at position 112 was used (AEDANS-apoE3/W@264). Fluorescence quenching studies revealed a solvent-exposed location for Trp-264. In the lipid-free state, fluorescence resonance energy transfer (FRET) was noted between Trp-264 and AEDANS, with a calculated distance of 27 A between the two fluorophores. Control experiments established that FRET observed in this system is intramolecular. FRET was abolished upon proteolysis in the linker region connecting the NT and CT domains. Lowering the solution pH to 4 induced an increase in the efficiency of intramolecular energy transfer, with the two domains reorienting about 5 A closer to one another. Interdomain FRET was retained in the presence of 0.6-1.0 m guanidine hydrochloride but was lost at higher concentrations, a manifestation of unfolding of the domains and increased distance between the donor-acceptor pair. Interaction of AEDANS-apoE3/W@264 with lipid induced a loss of FRET, attributed to spatial repositioning of the domains by >80 A. The data provide biophysical evidence that, in addition to reported conformational changes in the four-helix bundle configuration induced by lipid association, lipid binding of apoE is accompanied by reorientation of the tertiary disposition of the NT and CT domains.     

Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4

Human apolipoprotein (apo) E4 binds preferentially to very low-density lipoproteins (VLDLs), whereas apoE3 binds preferentially to high-density lipoproteins (HDLs), resulting in different plasma cholesterol levels for the two isoforms. To understand the molecular basis for this effect, we engineered the isolated apoE N-terminal domain (residues 1-191) and C-terminal domain (residues 192-299) together with a series of variants containing deletions in the C-terminal domain and assessed their lipid and lipoprotein binding properties. Both isoforms can bind to a phospholipid (PL)-stabilized triolein emulsion, and residues 261-299 are primarily responsible for this activity. ApoE4 exhibits better lipid binding ability than apoE3 as a consequence of a rearrangement involving the segment spanning residues 261-272 in the C-terminal domain. The strong lipid binding ability of apoE4 coupled with the VLDL particle surface being ～60% PL-covered is the basis for its preference for binding VLDL rather than HDL. ApoE4 binds much more strongly than apoE3 to VLDL but less strongly than apoE3 to HDL(3), consistent with apoE-lipid interactions being relatively unimportant for binding to HDL. The preference of apoE3 for binding to HDL(3) arises because binding is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover ～80% of the HDL(3) particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL(3) and VLDL is dependent upon two factors: (1) the stronger lipid binding ability of apoE4 relative to that of apoE3 and (2) the differences in the nature of the surfaces of VLDL and HDL(3) particles, with the former being largely covered with PL and the latter with protein.