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To understand the molecular basis for the differences in receptor-binding activity of the three common human apolipoprotein E (apoE) isoforms, we characterized the microenvironments of their LDL receptor (LDLR)-binding regions (residues 136;-150). When present in dimyristoyl phosphatidylcholine (DMPC) complexes, the 22-kDa amino-terminal fragments (residues 1;-191) of apoE3 and apoE4 bound to the LDLR with approximately 100-fold greater affinity than the 22-kDa fragment of apoE2. The pK(a) values of lysines (K) at positions 143 and 146 in the LDLR-binding region in DMPC-associated 22-kDa apoE fragments were 9.4 and 9.9 in apoE2, 9.5 and 9.2 in apoE3, and 9.9 and 9.4 in apoE4, respectively. The increased pK(a) of K146 in apoE2 relative to apoE3 arises from a reduction in the positive electrostatic potential in its microenvironment. This effect occurs because C158 in apoE2, unlike R158 in apoE3, rearranges the intrahelical salt bridges along the polar face of the amphipathic alpha-helix spanning the LDLR-binding region, reducing the effect of the R150 positive charge on K146 and concomitantly decreasing LDLR-binding affinity.The C112R mutation in apoE4 that differentiates it from apoE3 did not perturb the pK(a) of K146 significantly, but it increased the pK(a) of K143 in apoE4 by 0.4 pH unit. This change did not alter LDLR-binding affinity. Therefore, maintaining the appropriate positive charge at the C-terminal end of the receptor-binding region is particularly critical for effective interaction with acidic residues on the LDLR.

Apolipoprotein E7 (apoE7) (apoE3 E244K/E245K) is a naturally occurring mutant in humans that is associated with increased plasma lipid levels and accelerated atherosclerosis. It is reported to display defective binding to low density lipoprotein (LDL) receptors, high affinity binding for heparin, and like apoE4, preferential association with very low density lipoproteins (VLDL). There are two potential explanations for the preference of apoE7 for VLDL: lysine mutations, which occur in the major lipid-binding region (residues 244-272) of the carboxy-terminal domain of apoE7, could either directly determine the lipoprotein-binding preference or could interact with negatively charged residues in the amino-terminal domain, resulting in a domain interaction similar to that in apoE4 (interaction of Arg-61 with Glu-255), which is responsible for the apoE4 VLDL preference. To distinguish between these possibilities, we determined the binding preferences of recombinant apoE7 and two amino-terminal domain mutants, apoE7 (E49Q/E50Q) and apoE7 (D65N/E66Q), to VLDL-like emulsion particles. ApoE7 and both mutants displayed a higher preference for the emulsion particles than did apoE3, indicating that the carboxy-terminal lysine mutations in apoE7 are directly responsible for its preference for VLDL. Supporting this conclusion, the carboxy-terminal domain 12-kDa fragment of apoE7 (residues 192;-299) displayed a higher preference for VLDL emulsions than did the wild-type fragment. In addition, lipid-free apoE7 had a higher affinity for heparin than did apoE. However, when apoE7 was complexed with dimyristoylphosphatidylcholine or VLDL emulsions, the affinity difference was eliminated. In contrast to previous studies, we found that apoE7 does not bind defectively to the LDL receptor, as determined in both cell culture and solid-phase assays.We conclude that the two additional lysine residues in the carboxy-terminal domain of apoE7 directly alter its lipid- and heparin-binding affinities. These characteristics of apoE7 could contribute to its association with increased plasma lipid levels and atherosclerosis.

Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

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

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.

Hydrodynamic, chromatographic, and spectroscopic techniques were used to study the aqueous solution properties of the two structural domains of human apolipoprotein (apo) E3. An amino-terminal thrombolytic fragment of apoE (22 kDa, residues 1-191) and a carboxyl-terminal thrombolytic fragment of apoE (10 kDa, residues 216-299) were used as models for the two domains. Sedimentation equilibrium ultracentrifugation showed that apoE and the 10-kDa model domain self-associated predominantly as tetramers. The 22-kDa model domain was primarily monomeric. Molecular weights calculated from the weight average sedimentation and diffusion coefficients or from the sedimentation coefficients and Stokes radii were in agreement with the sedimentation equilibrium results. Derived frictional coefficients suggest larger axial ratios and/or more extensive hydration for the apoE and the 10-kDa domain tetramers as compared with the 22-kDa domain. Proteolysis of apoE followed by high performance liquid chromatography showed rapid production of free 22-kDa domain, whereas the free 10-kDa domain appeared as a tetramer late in the course of the hydrolysis. Assessment by circular dichroism demonstrated that both model domains and apoE had over 54% alpha-helical content, which changed little in a detergent (octyl-beta-D-glucopyranoside) or lipid (dimyristoylphosphatidylcholine) environment. In contrast to the circular dichroism results, apoE and the 10-kDa domain showed a marked blue shift in the fluorescence maximum in a lipid environment. The results suggest that the self-association of apoE in solution as a tetramer is mediated by the carboxyl-terminal domain and that the amino- and carboxyl-terminal domains do not associate with one another. The amino-terminal domain is most likely compact and globular, whereas the carboxyl-terminal domain is probably elongated. The isolated model domains appear to have structures that are similar to those of the domains in the intact protein.

Apolipoprotein (apo) E3-Leiden is a variant of apoE that is associated with dominant expression of type III hyperlipoproteinemia and that is defective in binding to the low density lipoprotein receptor. Therefore, the structure of apoE3-Leiden was investigated. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis apoE3-Leiden and its 22-kDa amino-terminal thrombolytic fragment migrated with a higher than normal apparent molecular weight. The structural abnormality of apoE3-Leiden was determined by sequencing its CNBr-, tryptic-, and Staphylococcus aureusV8 protease-generated peptides. In contrast to normal apoE3, which has a cysteine at residue 112, apoE3-Leiden does not contain any cysteine and has an arginine at position 112 (as does apoE4, which also completely lacks cysteine). The basis for the molecular weight difference was determined to be a seven-amino acid insertion that is a tandem repeat of residues 121-127 of normal apoE3, i.e.Glu-Val-Gln-Ala-Met-Leu-Gly, resulting in apoE3-Leiden having 306 amino acids rather than 299. The negatively charged glutamyl residues within the insertion compensates for the arginine substitution at residue 112; thus apoE3-Leiden focuses in the E3 position. The low density lipoprotein receptor binding activities of both intact apoE3-Leiden and its 22-kDa thrombolytic fragment were determined in an in vitroassay. Although apoE3-Leiden had only about 25% of normal binding activity, its 22-kDa thrombolytic fragment had nearly normal binding, suggesting that the carboxyl-terminal domain of apoE3-Leiden modulates the receptor binding function of its amino-terminal domain.

An apolipoprotein (apo) A-I variant that has a relative charge of -1 compared to normal apo-A-I on isoelectric focusing gels has been identified in five unrelated families as a result of screening a large number of individuals. The cause of the electrophoretic abnormality has been examined by analyzing the variant apo-A-I structure. The evidence suggests that a single amino acid, lysine 107, has been deleted in the variant apo-A-I of all affected individuals studied from these families, with the remainder of the variant apo-A-I sequence being unaffected. The deletion of this single basic amino acid residue is sufficient to account for the charge difference between the variant and normal apo-A-I as seen on isoelectric focusing gels. This variant, previously referred to as A-I-Marburg or A-I-Münster-2, can now be designated by the structural abnormality apo-A-I(Lys107—-0). The evidence from extensive pedigree analysis suggests the likelihood that the deletion mutant gene is allelic to the normal apo-A-I gene. At the same time, the kindred analyses have failed to yield a lipid abnormality that can be unequivocally related to the presence of this deletion mutant of apo-A-I. However, all subjects expressing apo-A-I(Lys107—-0) also express normal apo-A-I, so that any abnormality caused by the variant apo-A-I might be adequately compensated for by the normal apo-A-I. To examine directly the functional consequence of the lysine deletion, the isolated variant was tested in vitro for its ability to activate lecithin:cholesterol acyltransferase, the principal cholesterol-esterifying enzyme in plasma. It was found that apo-A-I(Lys107—-0) is deficient in its ability to activate lecithin:cholesterol acyltransferase, having only 40-60% of the cofactor activity of normal apo-A-I. The cofactor activity of the pro-apo-A-I component of the variant was also reduced to about 60% of either normal A-I or normal pro-apo-A-I. The functional defect is probably related to a disruption in the secondary and/or tertiary structure of the protein caused by the deletion of lysine 107 in the primary structure.

The low density lipoprotein (LDL) receptor plays a key role in cholesterol homeostasis, mediating cellular uptake of lipoprotein particles by high affinity binding to its ligands, apolipoprotein (apo) B-100 and apoE. The ligand-binding domain of the LDL receptor contains 7 cysteine-rich repeats of approximately 40 amino acids; each repeat contains 6 cysteines, which form 3 intra-repeat disulfide bonds. As a first step toward determining the structure of the LDL receptor, both free and bound to its ligands, we produced in Escherichia coli a soluble fragment containing the ligand-binding domain (residues 1-292) as a thrombin-cleavable, heat-stable thioredoxin fusion. Modest amounts (5 mg/liter) of partially purified but inactive fragment were obtained after cell lysis, heat treatment, thrombin cleavage, and gel filtration under denaturing conditions. We were able to refold the receptor fragment to an active conformation with approximately 10% efficiency. The active fragment was isolated and purified with an LDL affinity column. The refolded receptor fragment was homogeneous, as determined by sodium dodecyl sulfate or non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing. The purified fragment did not react with fluorescein-5-maleimide, indicating that all 42 cysteines were disulfide linked. In addition, the refolded fragment exhibited properties identical to those of the intact native receptor: Ca2+-dependent binding and isoform-dependent apoE binding (apoE2 binding <5% of apoE3). Furthermore, antibodies to the fragment recognized native receptors and inhibited the binding of 125I-LDL to fibroblast LDL receptors. We conclude that we have produced a properly folded and fully active receptor fragment that can be used for further structural studies.

Human apolipoprotein (apo) E contains an amino- and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoform-specific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186-202 or 186-223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction.