Structural variation in human apolipoprotein E3 and E4: secondary structure, tertiary structure, and size distribution

Human apolipoprotein E (apoE) is a 299-amino-acid protein with a molecular weight of 34 kDa. The difference between the apoE3 and apoE4 isoforms is a single residue substitution involving a Cys-Arg replacement at residue 112. ApoE4 is positively associated with atherosclerosis and late-onset and sporadic Alzheimer's disease (AD). ApoE4 and its C-terminal truncated fragments have been found in the senile plaques and neurofibrillary tangles in the brain of AD patients. However, detail structural information regarding isoform and domain interaction remains poorly understood. We prepared full-length, N-, and C-terminal truncated apoE3 and apoE4 proteins and studied their structural variation. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation revealed apoE3(72-299) as consisting of a major species with a sedimentation coefficient of 5.9. ApoE4(72-299) showed a wider and more complicated species distribution. Both apoE3 and E4 N-terminal domain (1-191) existed with monomers as the major component together with some tetramer. The oligomerization and aggregation of apoE protein increased when the C-terminal domain (192-271) was incorporated. The structural influence of the C-terminal domain on apoE is to assist self-association with no significant isoform preference. Circular dichroism and fluorescence studies demonstrated that apoE4(72-299) possessed a more alpha-helical structure with more hydrophobic residue exposure. The structural variation of the N-terminal truncated apoE3 and apoE4 protein provides useful information that helps to explain the greater aggregation of the apoE4 isoform and thus has implication for the involvement of apoE4 in AD.     

A monomeric, biologically active, full-length human apolipoprotein E

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.     

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.     

Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimer's disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.     

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.     

A segmental labeling strategy for unambiguous determination of domain–domain interactions of large multi-domain proteins

NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain–domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain–domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain–domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was ²H(99%)/¹⁵N-labeled whereas the C-terminal (CT-) domain was either ¹⁵N- or ¹⁵N/¹³C-labeled. 3-D ¹⁵N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.     

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.     

Structural and functional variations in human apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.     

Effects of polymorphism on the lipid interaction of human apolipoprotein E

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

Role of the N- and C-terminal domains in binding of apolipoprotein E isoforms to heparan sulfate and dermatan sulfate: a surface plasmon resonance study

The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.     

Apolipoprotein E and Low-Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform-Specific Alterations

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ? E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human ¹²⁵I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a K_D of 1.8 nM and a B_max of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade ¹²⁵I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with ¹²⁵I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. ¹²⁵I-ApoE2 binding was significantly lower than that of the other ¹²⁵I-apoE isoforms in both cell types. ¹²⁵I-ApoE4 binding was similar to that of ¹²⁵I-apoE3 in both astrocytes and neurons. On the other hand, ¹²⁵I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.     

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.     

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.     

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.     

Characterization of the heparin binding sites in human apolipoprotein E

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK(a) values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.     

A complete backbone spectral assignment of lipid-free human apolipoprotein E (apoE)

Apolipoprotein E is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer’s disease, cognitive function, immunoregulation, cell signaling and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, there is no structure available for the apoE C-terminal domain and full-length apoE. Here we report a complete NMR backbone spectral assignment of lipid-free human apoE.     

Apolipoprotein E4 domain interaction occurs in living neuronal cells as determined by fluorescence resonance energy transfer

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer disease. Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine whether apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N terminus, and cyan fluorescent protein (CFP) was fused to the C terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N and C termini must be in close proximity (<100 A). FRET signals occurred in cells transfected with YFP-apoE4-CFP but not in those transfected with YFP-apoE3-CFP, suggesting that the N and C termini of apoE4 are in close proximity in living cells and that those of apoE3 are not. FRET signals did not occur in cells cotransfected with YFP-apoE4 and apoE4-CFP, suggesting that the FRET in YFP-apoE4-CFP-transfected cells was intramolecular. Mutation of Arg-61 to Thr or Glu-255 to Ala in apoE4, which disrupts domain interaction, abolished FRET in Neuro-2a cells, strongly suggesting that the FRET in YFP-apoE4-CFP cells was caused by domain interaction. ApoE4-producing cells secreted less phospholipid than apoE3-producing cells, but after disruption of domain interaction in apoE4, phospholipid secretion increased to the levels seen with apoE3, suggesting that domain interaction decreases the phospholipid-binding capacity of apoE4. Thus, apoE4 domain interaction occurs in living neuronal cells and may be a molecular basis for apoE4-related neurodegeneration.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

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.     

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4相似文献