Lipid-bound Form Research Articles

Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs.

Serum amyloid A (SAA) is a highly conserved acute-phase protein that plays roles in activating multiple pro-inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high-density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein-protein interaction sites on SAA. While high-resolution structures for lipid-free, or apo-, forms of SAA have been reported, their relationship with the HDL-bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC-MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid-free and lipid-bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring-like structures reported for lipid-free ApoA-I. These SAA octamers represent a previously uncharacterized structure for lipid-free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA-I. The SAA-lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid-free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid-containing particles such as HDL. This study suggests a new model for SAA-lipid interactions and provides new insight into how SAA might stabilize protein-lipid nanodiscs or even replace ApoA-I as a scaffold for HDL particles during inflammation.

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein.

Apolipophorin III (apoLp-III) is an insect apolipoprotein that is predominantly present in a lipid-free state in the hemolymph. ApoLp-III from Galleria mellonella is able to interact with membrane components of Gram-negative bacteria, as part of an innate immune response to infection. The protein also exists in a lipoprotein-associated state when large amounts of lipids are mobilized. Therefore, lipid-bound apoLp-III was generated to analyze the binding interaction with lipopolysaccharides and phosphatidylglycerol, both abundantly present in membranes of Gram-negative bacteria. G. mellonella apoLp-III was lipidated with palmitoyl-2-oleoyl-glycero-3-phosphocholine to form lipid-protein complexes. The particle shape was discoidal with a 16.4nm diameter, a molecular mass of 460kDa, and contained 4 apoLp-III molecules. These discoidal lipoproteins were used to compare the lipopolysaccharide and phosphatidylglycerol binding activity with lipid-free apoLp-III. Lipopolysaccharide binding interaction was analyzed by non-denaturing PAGE, showing reduced ability of the lipid-bound protein to form lipopolysaccharide-protein complexes and to disaggregate lipopolysaccharide micelles. The apoLp-III-induced release of calcein from phosphatidylglycerol vesicles was decreased approximately fivefold when the protein was in the lipid-bound form, indicating reduced binding interaction with the phosphatidylglycerol membrane surface. These results show that when apoLp-III adopts a lipid-bound conformation, it is markedly less effective in interacting with lipopolysaccharides and phosphatidylglycerol vesicles. Thus, in order to be an effective antimicrobial protein, apoLp-III needs to be in a lipid-free state.

A Link Between Alzheimer's and Type II Diabetes Mellitus? Ca+2 -Mediated Signal Control and Protein Localization.

We propose protein localization dependent signal activation (PLDSA) as a model to describe pre-existing protein partitioning between the cytosol, and membrane surface, as a means to modulate signal activation, specificity, and robustness. We apply PLDSA to explain possible molecular links between type II diabetes mellitus (T2DM) and Alzheimer's disease (AD) by describing Ca+2 -mediated interactions between the Src non-receptor tyrosine kinase and p52Shc adaptor protein. We suggest that these interactions may serve as a contributing factor to disease development and progression. In particular, we propose that signaling response is regulated, in part, by Ca+2 -mediated partitioning of lipid-bound and soluble forms of Src and p52shc. Thus, protein-protein interactions that drive signaling in response to extracellular ligand binding are also mediated by partitioning of signaling proteins between membrane-bound and soluble populations. We propose that PLDSA effects may explain, in part, the evolutionary basis of promiscuous protein interaction domains and their importance in cellular function.

Structural Insight into Substrate Selection and Catalysis of Lipid Phosphate Phosphatase PgpB in the Cell Membrane

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro Here we report the crystal structure of the lipid-bound form of PgpB. The structure shows that a PE molecule is stabilized in a membrane-embedded tunnel formed by TM3 and the "PSGH" fingerprint peptide near the catalytic site, providing structural insight into PgpB substrate binding mechanism. Noteworthy, in silico docking of varied lipid phosphates exhibited similar substrate binding modes to that of PE, and the residues in the lipid tunnel appear to be important for PgpB catalysis. The catalytic triad in the active site is essential for dephosphorylating substrates lysophosphatidic acid, phosphatidic acid, or sphingosine-1-phosphate but surprisingly not for the native substrate PGP. Remarkably, residue His-207 alone is sufficient to hydrolyze PGP, indicating a specific catalytic mechanism for PgpB in PG biosynthesis. We also identified two novel sensor residues, Lys-93 and Lys-97, on TM3. Our data show that Lys-97 is essential for the recognition of lyso-form substrates. Modification at the Lys-93 position may alter substrate specificity of lipid phosphate phosphatase proteins in prokaryotes versus eukaryotes. These studies reveal new mechanisms of lipid substrate selection and catalysis by PgpB and suggest that the enzyme rests in a PE-stabilized state in the bilayer.

The antiapoptotic gene bcl-2 prevents reactivation of the senescence program induced by the histone deacetylase inhibitor sodium butyrate in rat fibroblasts transformed by the oncogenes E1A and c-Ha-Ras

The role of the gene bcl-2 conferring resistance to apoptosis in the activation of the cellular senescence program induced by the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBut) has been studied in transformed rat fibroblasts. The work was carried out in the rat embryo fibroblast cell line transformed by the oncogenes E1A, cHa-Ras, and bcl-2 (ERasBcl cells) resistant to apoptosis. The parent line of E1A-and cHa-Ras-transformed cells (ERas cells) was used as a control. It has been shown that NaBut suppresses the proliferation of ERasBcl cells to a much lesser extent than do the control ERas cells, in spite of the delay in cell cycle progression in the G1 phase for both lines. NaBut-induced hypertrophy is less marked in ERasBcl cells than in ERas cells due to the lower level of activation of the mTORC1 complex controlling protein synthesis and ribosome biogenesis. The activation of mTORC1 was assessed from the phosphorylation of its targets: ribosomal protein S6 and the inhibitor of translation initiation factor eIF4E (4E-BP1). Investigation of the dynamics of the level of one of the major markers of autophagosome formation (protein LC3) has shown the transient character of NaBut-induced accumulation of lipid-bound form LC3-II marking autophagosome membranes in bcl-2-expressing transformants in contrast to the ERas line. In addition, in ERasBcl cells there was no activation of the senescence-associated β-galactosidase (the marker for senescent cells). The results obtained suggest that the high level of expression of bcl-2, which blocks apoptotic cell death, prevents the implementation of the NaBut-induced tumor-suppressive cellular senescence program.

Structural basis for self-assembly of a cytolytic pore lined by protein and lipid.

Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a α-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

Membrane Partitioning of the Pore-Forming Domain of Colicin A. Role of the Hydrophobic Helical Hairpin

The colicins are bacteriocins that target Escherichia coli and kill bacterial cells through different mechanisms. Colicin A forms ion channels in the inner membranes of nonimmune bacteria. This activity resides exclusively in its C-terminal fragment (residues 387–592). The soluble free form of this domain is a 10 α-helix bundle. The hydrophobic helical hairpin, H8–H9, is buried inside the structure and shielded by eight amphipathic surface helices. The interaction of the C-terminal colicin A domain and several chimeric variants with lipidic vesicles was examined here by isothermal titration calorimetry. In the mutant constructions, natural sequences of the hydrophobic helices H8 and H9 were either removed or substituted by polyalanine or polyleucine. All the constructions fully associated with DOPG liposomes including the mutant that lacked helices H8 and H9, indicating that amphipathic rather than hydrophobic helices were the major determinants of the exothermic binding reactions. Alanine is not specially favored in the lipid-bound form; the chimeric construct with polyalanine produced lower enthalpy gain. On the other hand, the large negative heat capacities associated with partitioning, a characteristic feature of the hydrophobic effect, were found to be dependent on the sequence hydrophobicity of helices H8 and H9.

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe225) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL3) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe229 and Tyr192 residues were the main cleavage sites. Interestingly, the Phe225 residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL3; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL3 at only the N-terminus, especially at Phe33. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe225 and Phe229 residues newly exposed by chymase, but did not cleave Tyr192. These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

An optimized negative-staining protocol of electron microscopy for apoE4•POPC lipoprotein

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE•phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4•palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4•POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

Decoding of Lipoprotein−Receptor Interactions: Properties of Ligand Binding Modules Governing Interactions with Apolipoprotein E

Clusters of complement-type ligand binding repeats in the LDL receptor family are thought to mediate the interactions between these receptors and their various ligands. Apolipoprotein E, a key ligand for cholesterol homeostasis, has been shown to interact with LDLR, LRP, and VLDLR, through these clusters. LDLR and VLDLR each contain a single ligand binding repeat cluster, whereas LRP contains three large clusters of ligand binding repeats, each with ligand binding functions. We show that within sLRP3 the three-repeat subcluster CR16-18 recapitulated ligand binding to the isolated receptor binding portion of ApoE (residues 130-149). Binding experiments with LA3-5 of LDLR and CR16-18 showed that a conserved W25/D30 pair appears to be critical for high-affinity binding to ApoE(130-149). The triple repeat LA3-5 showed the expected interaction with ApoE(1-191).DMPC, but surprisingly CR16-18 did not interact with this form of ApoE. To understand these differences in ApoE binding affinity, we introduced mutations of conserved residues from LA5 into CR18 and produced a CR16-18 variant capable of binding ApoE(1-191).DMPC. This change cannot fully be accounted for by the interaction with the proposed ApoE receptor binding region; therefore, we speculate that LA5 is recognizing a distinct epitope on ApoE that may only exist in the lipid-bound form. The combination of avidity effects with this distinct recognition process likely governs the ApoE-LDL receptor interaction.

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3-phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P-enriched membranes and membrane-mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates approximately 24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P-containing membranes in vitro and in vivo, indicating that pH-dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH-sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form, promoting its penetration and increasing the membrane residence time.

Cleavage and functional loss of human apolipoprotein E by digestion of matrix metalloproteinase‐14

By means of a degradomic approach applying proteomic techniques, we previously suggested that apolipoprotein E (apoE) is a substrate of matrix metalloproteinase-14 (MMP-14). Here we confirm that apoE is, in fact, a substrate of MMP-14 and also of MMP-7 and MMP-2 to a lesser extent. The 34 kDa apoE protein was initially processed by MMP-14 into fragments with molecular masses of 28, 23, 21, and 11 kDa. MMP-14 cleavage sites within the apoE protein were determined by C-terminal labeling of MMP-14-digested apoE fragments with isotope ((18)O/(16)O = 1:1) and identification of the doublet fragments or peptides showing 2 Da difference by MS, along with N-terminal sequencing of the fragments. It was determined that the primary MMP-14 cleavage sites were A(176)-I(177), P(183)-L(184), P(202)-L(203), and Q(249)-I(250). The MMP-14-mediated cleavage of apoE was consistent regardless of whether apoE existed in its lipid-bound or lipid-free form. Upon digestion with MMP-14, apoE loses its ability to suppress the platelet-derived growth factor-induced migration of rat vascular smooth muscle cells. Considering the important role of apoE for lipid metabolism and atherosclerosis protection, our findings suggest that MMP-14 plays an essential role for the development of hyperlipidemia and atherosclerosis as a result of degradation of apoE.

Analysis of the Structural and Dynamic Properties of Human N-Terminal Domain of Apolipoprotein E by Molecular Dynamics Simulations

Whereas the lipid-free N-terminal domain of apolipoprotein E (apoE-NT) adopts a four-helix bundle, the lipid-bound form is believed to undergo a large conformational change likely to be characterized by the opening of the bundle. ApoE-NT in a water/alcohol mixture was also shown to experience conformational changes exhibiting similarities with those induced upon lipid binding. The structure and dynamics of apoE-NT have been here investigated by analyzing 40 ns and 60 ns molecular dynamics simulations performed in water and in a water/propanol mixture, respectively. The overall structural properties show alterations of the tertiary structure of apoE-NT in the water/alcohol system in agreement with those observed experimentally. In contrast, in the water simulation, the sampled conformations remain closer to the crystal structure that served as a starting point for both simulations. Interestingly, several propanol molecules are seen to penetrate two hydrophobic regions of the bundle interior. One of these regions is enclosed in part by the short helix (H1') connecting helices 1 and 2 of the bundle which has been experimentally shown to be important for modulating lipid binding activity of apoE-NT. Principal component analysis of the water/propanol trajectory confirms that the region including H1' is the locus of the largest motion. Another region involves the loop connecting helix 2 and helix 3 which has been hypothesized to play the role of a hinge in the opening of the bundle.

Avian apolipoprotein A-V binds to LDL receptor gene family members

Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits.

Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely, T cell receptor zetacyt, CD3epsiloncyt, CD3deltacyt, and CD3gammacyt, B cell receptor Igalphacyt and Igbetacyt, and Fc receptor FcepsilonRIgammacyt, using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that zetacyt, CD3epsiloncyt, and FcepsilonRIgammacyt bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of zetacyt and FcepsilonRIgammacyt does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

Apolipoprotein structural organization in high density lipoproteins: belts, bundles, hinges and hairpins

To summarize recent advances towards an understanding of the three-dimensional structures of the apolipoprotein components of HDL with a specific focus on high resolution models of apolipoprotein A-I. Since the primary sequence was first reported, various models have been advanced for the structure of apolipoprotein A-I, the major protein constituent of HDL, in its lipid-free and lipid-bound forms. Unfortunately, the generation of experimental data capable of distinguishing among the competing models has lagged far behind. However, recent experimental strategies, including X-ray crystallography, applications of resonance energy transfer and mass spectrometry, have combined with sophisticated theoretical approaches to develop three-dimensional structural models of apolipoprotein A-I with previously unavailable resolution. The recent synergy of sophisticated computer modeling techniques with hard experimental data has generated new models for apolipoprotein A-I in certain subclasses of HDL produced in vitro. The challenge now is to adapt and test these models in the more complex forms of HDL isolated directly from human plasma.

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

Apolipoprotein E (apoE) is a 34-kDa resident of lipoproteins that plays a key role in cholesterol homeostasis in plasma and in brain. It is composed of an N-terminal (NT) domain (residues 1-191) and a C-terminal (CT) domain (residues 201-299). Of the three major isoforms (apoE2, -E3, and -E4), apoE4 is considered a risk factor for both cardiovascular and Alzheimer disease. Compared with apoE3, domain interaction between NT and CT domains is believed to direct the lipoprotein distribution preference of apoE4 for very low density lipoprotein-sized particles. We examined the relative disposition of apoE4 NT and CT domains in lipid-free and lipid-bound forms by monitoring pyrene excimer fluorescence emission as a direct indicator of spatial proximity. Site-specific labeling of apoE4 by N-(1-pyrene)maleimide was accomplished after substitution of Cys residues for Arg-61 in NT domain and Glu-255 in CT domain. Pyrene labeling did not alter the lipoprotein distribution pattern of apoE4 in plasma. Pyrene excimer fluorescence was noted in lipid-free pyrene-R61C/E255C/apoE4 in mixtures containing excess wild-type apoE4, which was attributed to intramolecular spatial proximity between these specified sites. Upon disruption of tertiary interaction, a large decrease in excimer fluorescence emission was noted in pyrene-R61C/E255C/apoE4. In dimyristoylphosphatidylcholine/pyrene-R61C/E255C/apoE4 discoidal complexes, pyrene excimer fluorescence emission was retained. Taken together with fluorescence quenching and cross-linking analysis, a looped-back model of apoE4 is proposed in lipid-bound state, including spherical lipoprotein particles, wherein residues Arg-61 and Glu-255 are proximal to one another.