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.     

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

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

Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: correlation with protein stability?

Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of -helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon decreasing the pH to 3.0. Taken together, the data indicate that changes in the stability of secondary structure elements in apoE-NT isoforms are not responsible for pH-induced increases in lipid binding activity. It is likely that pH-induced disruption of inter-helical tertiary contacts may promote helix bundle conformational changes that present the hydrophobic interior of the protein to potential lipid surface binding sites.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.     

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

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

Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1–191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.     

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.     

Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.     

Molecular studies of pH-dependent ligand interactions with the low-density lipoprotein receptor

The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-null apoE3-NT. In binding experiments with wild-type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His --> Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization.     

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

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

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

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

Modification by acrolein, a component of tobacco smoke and age-related oxidative stress, mediates functional impairment of human apolipoprotein E

Oxidative damage to proteins such as apolipoprotein B-100 increases the atherogenicity of low-density lipoproteins (LDL). However, little is known about the potential oxidative damage to apolipoprotein E (apoE), an exchangeable antiatherogenic apolipoprotein. ApoE plays an integral role in lipoprotein metabolism by regulating the plasma cholesterol and triglyceride levels. Hepatic uptake of lipoproteins is facilitated by apoE's ability to bind with cell surface heparan sulfate proteoglycans and to lipoprotein receptors via basic residues in its 22 kDa N-terminal domain (NT). We investigated the effect of acrolein, an aldehydic product of endogenous lipid peroxidation and a tobacco smoke component, on the conformation and function of recombinant human apoE3-NT. Acrolein caused oxidative modification of apoE3-NT as detected by Western blot with acrolein-lysine-specific antibodies, and tertiary conformational alterations. Acrolein modification impairs the ability of apoE3-NT to interact with heparin and the LDL receptor. Furthermore, acrolein-modified apoE3-NT displayed a 5-fold decrease in its ability to interact with lipid surfaces. Our data indicate that acrolein disrupts the functional integrity of apoE3, which likely interferes with its role in regulating plasma cholesterol homeostasis. These observations have implications regarding the role of apoE in the pathogenesis of smoking- and oxidative stress-mediated cardiovascular and cerebrovascular diseases.     

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.     

Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors

Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.     

Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.     

Apolipoprotein E isoform-specific binding to the low-density lipoprotein receptor

Apolipoprotein E (apoE) is a ligand for members of the low-density lipoprotein receptor (LDLR) family and functions in plasma cholesterol homeostasis. A fluorescence-based assay has been employed in molecular studies of receptor-ligand interactions. Competition experiments revealed isoform-specific differences in binding of lipid-associated apoE N terminal (NT) domain to a recombinant soluble LDLR (sLDLR). In a similar manner, lipid--associated-but not lipid-free--full-length apoE3 showed binding activity to sLDLR. The molecular chaperone, receptor-associated protein, inhibited apoE3-NT-phospholipid complex binding to sLDLR. Kinetic studies of apoE3-NT-phospholipid complex interaction with sLDLR revealed time-dependent effects of apoE-NT isoform binding to sLDLR. The results reveal a discerning method for study of the molecular basis of ligand interactions that likely influence receptor function in maintenance of whole body cholesterol homeostasis.     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

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

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

apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

利用RT-PCR以及实时定量RT-PCR检测11个动脉粥样硬化(atherosclerosis,AS)相关基因在1、2和3月龄的载脂蛋白E(apolipoproteinE,aopE)/低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)双基因缺失(apoE-/-/LDLR-/-)小鼠主动脉中的表达变化,同时应用血生化指标和病理形态学观察AS早期病变特点,探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制.结果显示,apoE-/-/LDLR-/-小鼠IL-18、TLR2、MCP-1、ICAM-1、VCAM-1、GM-CSF、CD36和ET-1表达在1月龄时较同龄野生型(wild type,WT)小鼠显著上调(P<0.05,P<0.01),PDGF-α和TNF-α表达在2月龄时较同龄WT小鼠显著上调,除ET-1表达在2月龄时以及了LR2、VCAM-1和ICAM-1表达在3月龄时降至WT小鼠水平以外,其余各基因表达随年龄增长继续升高(P<0.05,P<0.01),其中MCP-1表达在2月龄时达到峰值.NF-kB在各年龄段apoE-/-/LDLR-/-小鼠中的表达与同龄WT小鼠相比均无显著差异.各年龄段apoE-/-/LDLR-/-/小鼠血清了C、TG、LDL、HDL、TNF-α、IL-1β和ox-LDL含量均显著高于同龄WT小鼠(P<0.05,P<0.01),并随年龄增长逐渐升高.apoE-/-/LDLR-/-小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积,随着年龄增长病变区域增多,脂质沉积增厚.上述结果提示:apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达,起始并扩大病变部位的炎症反应,共同促进AS的发生发展.